Process for preparing a compound useful to treat mycoses

ABSTRACT

A process for preparing a compound of formula (II): 
     
       
         
         
             
             
         
       
     
     includes reacting a compound of formula (VIII): 
     
       
         
         
             
             
         
       
     
     with a compound of formula (XIV):

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.16/569,128 filed on Sep. 12, 2019, which is a Continuation of U.S.application Ser. No. 16/422,062 filed on May 24, 2019, now U.S. Pat. No.10,450,304, issued Oct. 22, 2019, which is a Continuation of U.S.application Ser. No. 16/139,591 filed on Sep. 24, 2018, now U.S. Pat.No. 10,344,022, issued Jul. 9, 2019, which is a Continuation of U.S.application Ser. No. 15/524,505, filed on May 4, 2017, now U.S. Pat. No.10,106,531, issued Oct. 23, 2018, which is a National Phase of PCTInternational Application No. PCT/GB2015/053733 filed on Dec. 4, 2015,which claims priority under 35 U.S.C. § 119(a) to Patent Application No.14196662.2 filed in Europe on Dec. 5, 2014. All of the aboveapplications are hereby expressly incorporated by reference into thepresent application.

FIELD OF THE INVENTION

This invention relates to a compound useful in the treatment of mycoses,compositions containing it and its use in therapy.

BACKGROUND OF THE INVENTION

The incidence of fungal infections has increased substantially over thepast two decades and invasive forms are leading causes of morbidity andmortality, especially amongst immunocompromised or immunosuppressedpatients. Disseminated candidiasis, pulmonary aspergillosis, andemerging opportunistic fungi are the most common agents producing theseserious mycoses. It is a particular feature of fungi that they are ableto generate an extracellular matrix (ECM) that binds them together andallows them to adhere to their in vitro or in vivo substrates. Thesebiofilms serve to protect them against the hostile environments of thehost immune system and to resist antimicrobial killing (Kaur and Singh,2013).

Pulmonary aspergillosis can be segmented into those patients sufferingwith non-invasive disease versus those with an invasive condition. Afurther sub-division is used to characterise patients who exhibit anallergic component to aspergillosis (known as ABPA; allergicbronchopulmonary aspergillosis) compared with those that do not. Thefactors precipitating pulmonary aspergillosis may be acute, such asexposure to high doses of immuno-suppressive medicines or to intubationin an intensive care unit. Alternatively, they may be chronic, such as aprevious infection with TB (Denning et al., 2011a). Chronic lunginfections with aspergillus can leave patients with extensive andpermanent lung damage, requiring lifetime treatment with oral azoledrugs (Limper et al., 2011).

A growing body of research suggests that aspergillus infection may playan important role in clinical asthma (Chishimba et al., 2012;Pasqualotto et al., 2009). Furthermore, recently published work hascorrelated aspergillus infection with poorer clinical outcomes inpatients with COPD (Bafadhel et al., 2013). Similarly cross-sectionalstudies have shown associations between the presence of Aspergillus spp.and Candida spp. in the sputum and worsened lung function (Chotirmall etal., 2010; Agbetile et al., 2012).

Invasive aspergillosis (IA) exhibits high mortality rates inimmunocompromised patients, for example, those undergoing allogenic stemcell transplantation or solid organ transplants (such as lungtransplants). The first case of IA reported in an immunocompromisedpatient occurred in 1953. This event was concurrent with theintroduction of corticosteroids and cytotoxic chemotherapy intotreatment regimens (Rankin, 1953). Invasive aspergillosis is a majorconcern in the treatment of leukaemia and other haematologicalmalignancies given its high incidence and associated mortality. Deathrates usually exceed 50% (Lin et al., 2001) and long term rates canreach 90% in allogeneic hematopoietic stem cell transplantationrecipients, despite the availability of oral triazole medicines(Salmeron et al., 2012). In patients undergoing solid organtransplantation (particularly of the lung), the use of high doses ofsteroids leaves patients vulnerable to infection (Thompson andPatterson, 2008) which is a serious problem. The disease has alsoappeared in less severely immunocompromised patient populations. Theseinclude those suffering with underlying COPD or cirrhosis, patientsreceiving high dose steroids, and individuals fitted with central venouscatheters or supported by mechanical ventilation (Dimopoulos et al.,2012).

Existing anti-fungal medicines are predominantly dosed either orally orsystemically. These commonly exploited routes of delivery are poor fortreating lung airways infections, since drug concentrations achieved atthe site of infection tend to be lower than those in organs. This isespecially so for the liver, which is a site of toxicity: up to 15% ofpatients treated with voriconazole suffer raised transaminase levels(Levin et al., 2007; Lat and Thompson, 2011). Exposure of the liver alsoresults in significant drug interactions arising from the the inhibitionof hepatic P450 enzymes (Jeong, et al., 2009; Wexler et al., 2004).

Furthermore, the widespread use of triazoles, both in the clinic and inagriculture has led to a growing and problematic emergence of resistantmycoses in some locations (Denning et al., 2011b; Bowyer and Denning,2014).

It is clearly evident that an urgent medical need exists for novelanti-fungal medicines that deliver improved efficacy and better systemictolerability profiles.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides compounds of formula (I),

wherein:

-   -   R represents hydrogen, halo, cyano, C₁₋₄ haloalkyl, C₁₋₄        haloalkoxy, or SO₂NR⁵R⁶;    -   R^(1a) and R^(1b) independently represent hydrogen or halo;    -   R² represents hydrogen, halo, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy or        C₁₋₄ haloalkoxy;    -   R³ represents, halo, cyano, C₁₋₄ alkyl, or C₁₋₄ hydroxyalkyl;    -   R⁴ represents, hydrogen or C₁₋₄ alkyl;    -   X represents CH or N;    -   Y represents CH or N;    -   R⁵ and R⁶ independently represent hydrogen or C₁₋₄ alkyl

and pharmaceutically acceptable salts thereof (sometimes referred tohereinafter as “compounds of the invention” or “compounds of thedisclosure”).

Biological data disclosed herein below reveals that the compounds of theinvention are potent inhibitor of Aspergillus fumigatus growth in invitro assays. In immunosuppressed mice compounds of the inventiondemonstrated potent inhibition of Aspergillus fumigatus infections.Other desirable properties of compounds of the invention are describedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 displays the effects of prophylactic and therapeutic treatmentwith compound Example 1 on CFU in the lungs of Aspergillus fumigatusinfected, immuno-compromised, neutropenic mice.

FIG. 2 and FIG. 3 show the effects of prophylactic and therapeutictreatment with compound Example 1 on galactomannan concentrations inBALF and serum respectively, in Aspergillus fumigatus infected,immuno-compromised, neutropenic mice.

In FIGS. 1-3, the symbol *** indicates significance with P<0.001.

DETAILED DESCRIPTION OF THE INVENTION

Alkyl groups may be branched or straight chain. C₁₋₄ alkyl groups mayfor example represent C₁₋₃ alkyl or C₁₋₂ alkyl. Exemplary alkyl groupsinclude methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl andCH₂CHMe₂. In one embodiment alkyl refers to straight chain alkyl.

Alkoxy as used herein means —Oalkyl and includes straight or branchedchain alkoxy, for example methoxy, ethoxy, propoxy, butoxy.

Hydroxyalkyl means alkyl with a hydroxyl substituent in any position.Examples include hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl and4-hydroxy-n-butyl.

Halogens may suitably be Br, Cl or F, especially Cl or F, particularlyF.

In one embodiment there is provided a pharmaceutically acceptable saltof the compound of the invention.

The compounds of the disclosure include those wherein the atom specifiedis a naturally occurring or non-naturally occurring isotope. In oneembodiment the isotope is a stable isotope. Thus the compounds of thedisclosure include, for example those containing one or more deuteriumatoms in place of hydrogen atoms and the like.

The disclosure also extends to all polymorphic forms of the compoundsherein defined.

The disclosure also extends to all solvate of the compounds hereindefined. Examples of solvates include hydrates.

Suitably R represents H, F, CN, OCHF₂, OCF₃, SO₂NH₂, SO₂NHMe, SO₂NMe₂ orCl, especially F or CN, more preferably F.

Suitably R^(1a) represents H, F or Cl, especially H.

Suitably R^(1b) represents H or F, especially H.

Suitably R² represents H, Me or OMe, especially H.

Suitably R³ represents Me, CN, Cl, CH₂OH or F, especially Me.

Suitably R⁴ represents H or Me, especially H.

Suitably R⁵ represents H or Me.

Suitably R⁶ represents H or Me.

Suitably NR⁵R⁶ represents NH₂, NHMe or NMe₂.

Suitably X represents CH or N, especially CH.

Suitably Y represents CH (which carbon atom may optionally besubstituted by R, R^(1a), or R^(1b) for example by F) or N, especiallyCH.

Suitably R^(1a) and R^(1b) each represent H and R represents F, CN,OCHF₂, OCF₃, SO₂NH₂, SO₂NHMe, SO₂NMe₂, or Cl, especially R^(1a) andR^(1b) represent H and R represents F or CN, more preferably R^(1a) andR^(1b) represent H and R represents F.

Alternatively suitably R^(1a) represents F, R^(1b) represents H and Rrepresents F or OCHF₂, for example, R^(1a) represents F, R^(1b)represents H and R represents F.

Alternatively suitably R^(1a) represents Cl, R^(1b) represents H and Rrepresents F.

Suitably R⁴ represents H and R³ represents Me, CN, Cl, CH₂OH or F,especially R⁴ represents H and R³ represents Me.

Alternatively suitably R⁴ represents Me and R³ represents Me.

Suitably Y represents CH, R^(1a) and R^(1b) each represent H and R is inthe 3-position or 4-position, especially the 4-position.

Alternatively suitably Y represents CH, R^(1b) represents H and R andR^(1a) are in the 2,4-positions, 3,4-positions, 2,5-positions or3,5-positions, especially the 2,4-positions.

Alternatively suitably Y represents N, R^(1a) and R^(1b) each representH and R is in the 4-position, 5-position or 6-position, especially the5-position.

Suitably Y represents CH and R, R^(1a) and R^(1b) are in the 2,4 and,6-positions.

Suitably R² is located ortho to the nitrogen of the piperazinylsubstituent. Alternatively, R² is located meta to the nitrogen of thepiperazinyl substituent.

Suitably R⁴ is located ortho to the oxygen of the ether substituent.

Suitably the aromatic moiety comprising Y represents 4-fluorophenyl,3-fluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 4-cyanophenyl, 3-cyanophenyl,4-difluoromethoxyphenyl, 4-(trifluoromethoxy)phenyl, 4-sulfamoylphenyl,4-(N-methylsulfamoyl)phenyl, 4-(N-N-dimethylsulfamoyl)phenyl,4-cyano-2-fluorophenyl, 4-(difluoromethoxy)-3-fluorophenyl,4-chloro-2-fluorophenyl, 4-chloro-3-fluorophenyl, 2,4,6-trifluorophenyl,5-fluoropyridin-2-yl, 5-cyanopyridin-2-yl, 6-cyanopyridin-2-yl,4-cyanopyridin-2-yl, 5-(trifluoromethoxy)pyridin-2-yl or5-chloropyridin-2-yl, especially 4-fluorophenyl or 4-cyanophenyl, morepreferably 4-fluorophenyl.

In an embodiment, the compound of formula (I) is selected from:

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(2,4-difluorophenyl)benzamide;

4-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-cyanophenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-2,5-dimethylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(5-fluoropyridin-2-yl)benzamide;

4-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(5-cyanopyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(3-cyanophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-cyanophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-chlorophenyl)piperazin-1-yl)-N-(4-cyanophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3,5-dimethylphenyl)piperazin-1-yl)-N-(4-cyanophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-difluoromethoxyphenyl)benzamide;

4-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-(trifluoromethoxy)phenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)piperazin-1-yl)-N-(3-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3,5-dimethylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-hydroxymethylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-fluorophenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-chlorophenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-sulfamoylphenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-(N-methylsulfamoyl)phenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)piperazin-1-yl)-N-(4-(N,N-dimethylsulfamoylphenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-fluorophenyl)piperazin-1-yl)-N-(4-sulfamoylphenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-cyano-2-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-fluorophenyl)piperazin-1-yl)-N-(4-cyano-2-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-(difluoromethoxy)-3-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(2,5-difluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(3,4-difluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)piperazin-1-yl)-N-(3,5-difluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-chloro-2-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-chloro-3-fluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)piperazin-1-yl)-N-(2,4,6-trifluorophenyl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)-3-methylbenzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)-2-methylbenzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)-3-methoxybenzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)-2-methoxybenzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)piperazin-1-yl)-N-(6-cyanopyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-cyanopyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-hydroxymethylphenyl)piperazin-1-yl)-N-(5-cyanopyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(5-(trifluoromethoxy)pyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-fluorophenyl)piperazin-1-yl)-N-(5-fluoropyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(5-chloropyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)6-methylpyridin-2-yl)piperazin-1-yl)-N-(5-cyanopyridin-2-yl)benzamide;

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)piperazin-1-yl)-N-(5-fluoropyridin-2-yl)benzamide;

and pharmaceutically acceptable salts thereof.

In an embodiment, the compound of formula (I) is not:

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl) benzamide

or a pharmaceutically acceptable salt thereof.

The compounds of the invention may be prepared from commerciallyavailable starting materials by the non-limiting synthetic methodologiesdepicted below (Schemes 1-3).

Thus compounds of formula (I) may be obtained by a general process(Scheme 1) whereby a benzoic acid precursor (II), or a suitablyprotected derivative thereof, is reacted with an activating agent, togenerate a reactive, electrophilic carboxylic acid derivative, followedby subsequent reaction with an amine of formula (III), or a suitablyprotected derivative thereof. It will be understood by persons skilledin the art that, in some instances, the activated carboxylic acidderivative, such as an acid chloride, may be isolated or in other casesmay be a transient intermediate that is not isolated, but generated insitu and used directly. Reagents suitable for the activation of thecarboxylate group include carbonyl diimidazole,1-chloro-N,N,2-trimethylprop-1-en-1-amine and a wide selection ofpeptide coupling agents such asbenzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP®), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(EDCI.HCl) and the like. Such reactions are conveniently carried out ina non-polar, aprotic solvent, such as DCM or, in some cases, polar, nonprotic solvents such as pyridine. The resulting amides may be generatedat or below ambient temperature, such as RT or may be formed at elevatedtemperatures for example 60° C. A review of methodologies for thepreparation of amides is covered in: ‘Amide bond formation and peptidecoupling’ Montalbetti, C. A. G. N. and Falque, V. Tetrahedron, 2005, 61,10827-10852. The compounds of formula (I) are revealed, in thoseinstances wherein one or more protective groups have been employed byappropriate deprotection steps.

The intermediates of formula (II) may be derived by a noblemetal-mediated bond forming process, such as a Buchwald couplingreaction, between a piperazine derivative of formula (V) and a4-bromobenzoate of formula (VI) to provide, in the first instance, thecorresponding benzoic acid esters of formula (IV). Those skilled in theart will appreciate that a wide variety of conditions may be used foraffecting transformations of this kind. In particular, palladiumcatalysts and phosphine ligands such as RuPhosG3 and RuPhos areroutinely employed in the presence of a base, for example, cesiumcarbonate or lithium hexamethyldisilazide. Such coupling procedures arecommonly carried out in polar, non protic solvents such as DMF and atelevated temperatures, for example at 70-80° C. The compounds of formula(II) are obtained following subsequent hydrolysis of the esters (IV) tothe free acid. Conditions suitable for this functional groupinterconversion depend upon the nature of the ester. Primary andsecondary esters (for example R^(a)=Me, Et and Pr^(i)) are convenientlysaponified by exposure to a suitable inorganic base, for example lithiumhydroxide, in an aqueous mixture of aprotic and/or protic solvents, suchTHF:methanol:water. More hindered examples (for example R^(a)=^(t)Bu)may be more readily de-esterified by treatment with a strong mineralacid such as hydrochloric acid or a strong organic acid, typicallytrifluoroacetic acid, used either neat or in the presence of a solventsuch as DCM.

In an alternative process, using the Buchwald coupling methodology, theadvanced ester intermediates of formula (IV) are also available byreaction of the aryl bromides of formula (VIII) with the piperazinemotifs of formula (XIV) (R^(a)=lower alkyl, such as C₁₋₅ alkyl, forexample methyl or ethyl, or else tert-butyl) in a similar manner to thatdescribed above. The same technology may be applied to the aryl bromide(VIII) and a suitably protected piperazine of formula (IX), therebygenerating the intermediates of formula (VII), which are transformed tothe aforementioned compounds of formula (V) by an appropriatedeprotection step. A nitrogen protective group strategy, fit for thispurpose, is a urethane employing, for example, the Boc group (P═CO₂^(t)Bu) which is stable under the conditions required for the couplingreaction and may be removed thereafter by treatment with acid. Removalof an N-Boc protective group is typically achieved by treatment with astrong organic acid such as TFA in an inert solvent such DCM at ambienttemperature.

In a third approach the benzoic acid esters of formula (IV) may bederived by the reaction of the phenolic intermediates (XIII) [Y═CH] orthe corresponding pyridinols [Y═N] with an alkylating agent of formula(XI), wherein Z represents a leaving group and the stereochemistry ofthe said reagent is absolute as depicted. Typical alkylating agents fortransformations of this kind include sulfonate esters such as mesylates(Z=Me SO₂O) or triflates (Z═CF₃SO₂O) as well as alkyl halides, forexample the chloromethyl or the bromo-methyl derivative (Z═Cl and Brrespectively).

A significant practical consideration is that the tosylate derivative(XIa) [Z=p-TolylSO₂O], namely:((3S,5R)-5-((1H-1,2,4-triazol-1-yl)methyl-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methyl 4-methylbenzenesulfonate, is a widely available reagent, in bulk,from commercial sources, in high enantiomeric purity. This derivative isalso chemically and configurationally stable and, as a solid, offersoperational advantages over alternative, more volatile (and thereforemore hazardous) alkylating agents. The etherification process istypically carried out under basic conditions, for example in thepresence of sodium ethoxide (to generate the phenoxide anion in situ) ina polar, aprotic solvent, suitably DMF, and at temperatures in theregion of 40-50° C.

In a similar manner to that already described above, the 1,4-diarylatedpiperazines of formula (XIII) originate from the Buchwald coupling ofthe mono-substituted piperazines (XIV), with bromophenols of formula (X)[Y═CH, R^(c)═H] or with the corresponding bromo-pyridinols [Y═N,R^(c)═H] or, in some instances, with suitably protected derivativesthereof [R^(c)≠H]. In those cases wherein the compound of formula (X)has been protected (for example as a methyl or a benzyl ether) thedesired coupled product (XIII) is obtained following an appropriatedeprotection step (for example O-demethylation with BBr₃ orhydrogenolysis).

The hydroxylated aryl bromides of formula (X) are also converted by thesame methodology into the N-arylpiperazines of formula (XII) by acoupling reaction with a mono-protected piperazine of formula (IX). Acommonly employed amine protective group for such a purpose is the Bocgroup in which case the compound of formula (IX) is tert-butylpiperazine-1-carboxylate. It will be apparent to those skilled in theart that other nitrogen protective groups may be selected based uponconsiderations of orthogonality and operational efficiency. Alkylationof the free hydroxyl function in the N-arylpiperazines of formula (XII)with the tosylate (XIa), as described above, gives rise to theintermediates of formula (VII).

It will be appreciated from the preparative routes outlined above(Scheme 1) that in some instances it is advantageous to perform the sameor similar synthetic transformations in a different order, so as toimprove the overall efficiency of the processes and/or the quality ofthe materials obtained therefrom. In addition to those examples alreadydisclosed the hydroxylated aryl bromides of formula (X) may betransformed into the compounds of formula (VII) by conducting the twosteps, outlined above, in reverse the order. Treatment of thephenols/pyridinols of formula (X) with the tosylate (XIa) provides theether derivatives of formula (VIII), which have been converted underBuchwald coupling conditions into the intermediates of formula (VII), aspreviously described.

Additional strategies for preparing the compounds of the invention,using the synthetic technologies described above, are revealed below(Scheme 2) in which the same or closely related intermediates appearingabove (Scheme 1) are assembled in a different order.

Treatment of the aniline components (III) with the benzoyl chlorides(XIX) provides the benzanilide derivatives of formula (XVIII). Asalready noted such amidic products may be prepared from thecorresponding amine and benzoic acids directly using a wide variety ofactivating agents, including peptide coupling reagents, of which manyare available in the art. Subjecting these products to the Buchwaldcoupling reaction with a suitable mono-protected piperazine (IX), underthe agency of a catalyst, in the manner recorded above, gives rise tothe intermediates of formula (XVII). Piperazine derivatives that arestable under these bond forming conditions include urethanes such as theN-Boc derivative, though alternatives (such as a Cbz group) may beadvantageous in some cases. Removal of the amine protective group undersuitable conditions and a second palladium mediated N-arylationprocedure with an aromatic bromide of formula (X), or a protectedderivative thereof, furnishes the advanced intermediates of formula(XV).

In some instances this transformation may be accomplished usingsubstrates (X) in which the free hydroxyl group is present (i.e. Rc=H),In other cases it may be preferable to protect the said functionalityas, for example, an ether derivative, typically as a benzyl ether, whichmay be reverted to the free phenol or pyridinol by O-dealkylation underhydrogenolytic conditions Representative compounds of formula (I) arethen obtained by the subsequent alkylation of the hydroxyl group with areagent of formula (XI), most suitably with the tosylate (XIa).

Compounds of the invention wherein the benzamide is formed with a2-aminopyridine, (I) [Y═N], are accessible from the palladium mediatedcross coupling of a primary carboxamide of formula (XX) with anoptionally substituted, 2-bromopyridine substrate of formula (XXI).(Scheme 3). Typical reaction conditions for effecting the Buchwaldamidation of aryl halides include the use of a palladium catalyst, suchas tris(dibenzylideneacetone)dipalladium(0) in the presence of aphosphine ligand, commonly Xantphos and the like and under basicconditions, for example using cesium carbonate. It is usual to conductsuch reactions in a polar, aprotic solvent, typically DMF, at elevatedtemperatures such as 80-100° C. The primary benzamides (XX) are readilygenerated from the corresponding benzoic acids (II) by treating themwith a source of ammonia, conveniently ammonium chloride, under standardpeptide coupling conditions.

Protective groups and the means for their removal are described in“Protective Groups in Organic Synthesis”, by Theodora W. Greene andPeter G. M. Wuts, published by John Wiley & Sons Inc; 4th Rev Ed., 2006,ISBN-10: 0471697540. A review of methodologies for the preparation ofamides is covered in: “Amide bond formation and peptide coupling”Montalbetti, C. A. G. N. and Falque, V. Tetrahedron, 2005, 61,10827-10852.

Thus the invention also provides a process for preparing a compound offormula (I) or a pharmaceutically acceptable salt thereof whichcomprises reacting a compound of formula (II):

-   -   wherein:        -   R² represents hydrogen, halo, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy            or C₁₋₄ haloalkoxy;        -   R³ represents, halo, cyano, C₁₋₄ alkyl, or C₁₋₄            hydroxyalkyl;        -   R⁴ represents, hydrogen or C₁₋₄ alkyl;        -   X represents CH or N;

or an activated derivative thereof (such as an acid halide, e.g. an acidchloride or an acid anhydride); or a salt thereof;

with a compound of formula (III):

-   -   wherein:        -   R represents hydrogen, halo, cyano, C₁₋₄ haloalkyl, C₁₋₄            haloalkoxy, or SO₂NR⁵R⁶;        -   R^(1a) and R^(1b) independently represent hydrogen or halo;        -   R⁵ and R⁶ independently represent hydrogen or C₁₋₄ alkyl;            and        -   Y represents CH or N;        -   or a salt thereof.

The invention also provides a process for preparing a compound offormula (I) or a pharmaceutically acceptable salt thereof whichcomprises reacting a compound of formula (XV):

-   -   wherein:        -   R represents hydrogen, halo, cyano, C₁₋₄ haloalkyl, C₁₋₄            haloalkoxy, or SO₂NR⁵R⁶;        -   R^(1a) and R^(1b) independently represent hydrogen or halo;        -   R² represents hydrogen, halo, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy            or C₁₋₄ haloalkoxy;        -   R³ represents, halo, cyano, C₁₋₄ alkyl, or C₁₋₄            hydroxyalkyl;        -   R⁴ represents, hydrogen or C₁₋₄ alkyl;        -   R⁵ and R⁶ independently represent hydrogen or C₁₋₄ alkyl;        -   X represents CH or N; and        -   Y represents CH or N;

or a salt thereof;

with a compound of formula (XI):

-   -   wherein:        -   Z represents a leaving group such as p-TolylSO₂O;

or a salt thereof.

The invention also provides a process for preparing a compound offormula (I) or a pharmaceutically acceptable salt thereof whichcomprises reacting a compound of formula (XX):

-   -   wherein:        -   R² represents hydrogen, halo, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy            or C₁₋₄ haloalkoxy;        -   R³ represents, halo, cyano, C₁₋₄ alkyl, or C₁₋₄            hydroxyalkyl;        -   R⁴ represents, hydrogen or C₁₋₄ alkyl; and        -   X represents CH or N;

or a salt thereof;

with a compound of formula (XXI):

-   -   wherein:        -   R represents hydrogen, halo, cyano, C₁₋₄ haloalkyl, C₁₋₄            haloalkoxy, or SO₂NR⁵R⁶;        -   R^(1a) and R^(1b) independently represent hydrogen or halo;            and        -   R⁵ and R⁶ independently represent hydrogen or C₁₋₄ alkyl;

or a salt thereof.

Pharmaceutically acceptable salts of the compounds of formula (I)include in particular pharmaceutically acceptable acid addition salts ofsaid compounds. The pharmaceutically acceptable acid addition salts ofcompounds of formula (I) are meant to comprise the therapeuticallyactive non-toxic acid addition salts that the compounds of formula (I)are able to form. These pharmaceutically acceptable acid addition saltscan conveniently be obtained by treating the free base form with suchappropriate acids in a suitable solvent or mixture of solvents.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric acids and the like; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, malonic,succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic,ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-aminosalicylic, pamoic acid and the like.

Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The definition of the compounds of formula (I) is intended to includeall tautomers of said compounds.

The definition of the compounds of formula (I) is intended to includeall solvates of said compound (including solvates of salts of saidcompound) unless the context specifically indicates otherwise. Examplesof solvates include hydrates.

The compounds of the disclosure include embodiments wherein one or moreatoms specified are naturally occurring or non-naturally occurringisotopes. In one embodiment the isotope is a stable isotope. Thus thecompounds of the disclosure include, for example, deuterium containingcompounds and the like.

The disclosure also extends to all polymorphic forms of the compoundsherein defined.

Novel intermediates as described herein such as compounds of formula(II), (IV), (V), (VII), (VIII) (XIII), (XV) and (XX) and salts thereof,form a further aspect of the invention. Salts include pharmaceuticallyacceptable salts (such as those mentioned above) andnon-pharmaceutically acceptable salts. Salts of acids (e.g. carboxylicacids) include first and second group metal salts including sodium,potassium, magnesium and calcium salts.

In an embodiment there is provided a pharmaceutical compositioncomprising one or more compounds of the invention optionally incombination with one or more pharmaceutically acceptable diluents orcarriers.

Suitably the compounds of the invention are administered topically tothe lung or nose, particularly, topically to the lung. Thus, in anembodiment there is provided a pharmaceutical composition comprising oneor more compounds of the invention optionally in combination with one ormore topically acceptable diluents or carriers.

Suitable compositions for pulmonary or intranasal administration includepowders, liquid solutions, liquid suspensions, nasal drops comprisingsolutions or suspensions or pressurised or non-pressurised aerosols.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985). The compositions may also conveniently be administered inmultiple unit dosage form.

Topical administration to the nose or lung may be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.Such formulations may be administered by means of a nebuliser e.g. onethat can be hand-held and portable or for home or hospital use (i.e.non-portable). An example device is a RESPIMAT inhaler. The formulationmay comprise excipients such as water, buffers, tonicity adjustingagents, pH adjusting agents, viscosity modifiers, surfactants andco-solvents (such as ethanol). Suspension liquid and aerosolformulations (whether pressurised or unpressurised) will typicallycontain the compound of the invention in finely divided form, forexample with a D₅₀ of 0.5-10 μm e.g. around 1-5 μm. Particle sizedistributions may be represented using D₁₀, D₅₀ and D₉₀ values. The D₅₀median value of particle size distributions is defined as the particlesize in microns that divides the distribution in half. The measurementderived from laser diffraction is more accurately described as a volumedistribution, and consequently the D₅₀ value obtained using thisprocedure is more meaningfully referred to as a Dv₅₀ value (median for avolume distribution). As used herein Dv values refer to particle sizedistributions measured using laser diffraction. Similarly, D₁₀ and D₉₀values, used in the context of laser diffraction, are taken to mean Dv₁₀and Dv₉₀ values and refer to the particle size whereby 10% of thedistribution lies below the D₁₀ value, and 90% of the distribution liesbelow the D₉₀ value, respectively.

According to one specific aspect of the invention there is provided apharmaceutical composition comprising one or more compounds of theinvention in particulate form suspended in an aqueous medium. Theaqueous medium typically comprises water and one or more excipientsselected from buffers, tonicity adjusting agents, pH adjusting agents,viscosity modifiers and surfactants.

Topical administration to the nose or lung may also be achieved by useof an aerosol formulation. Aerosol formulations typically comprise theactive ingredient suspended or dissolved in a suitable aerosolpropellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon(HFC). Suitable CFC propellants include trichloromonofluoromethane(propellant 11), dichlorotetrafluoromethane (propellant 114), anddichlorodifluoromethane (propellant 12). Suitable HFC propellantsinclude tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227).The propellant typically comprises 40%-99.5% e.g. 40%-90% by weight ofthe total inhalation composition. The formulation may compriseexcipients including co-solvents (e.g. ethanol) and surfactants (e.g.lecithin, sorbitan trioleate and the like). Other possible excipientsinclude polyethylene glycol, polyvinylpyrrolidone, glycerine and thelike. Aerosol formulations are packaged in canisters and a suitable doseis delivered by means of a metering valve (e.g. as supplied by Bespak,Valois or 3M or alternatively by Aptar, Coster or Vari).

Topical administration to the lung may also be achieved by use of adry-powder formulation. A dry powder formulation will contain thecompound of the disclosure in finely divided form, typically with an MMDof 1-10 μm or a D₅₀ of 0.5-10 μm e.g. around 1-5 μm. Powders of thecompound of the invention in finely divided form may be prepared by amicronization process or similar size reduction process. Micronizationmay be performed using a jet mill such as those manufactured by HosokawaAlpine. The resultant particle size distribution may be measured usinglaser diffraction (e.g. with a Malvern Mastersizer 2000S instrument).The formulation will typically contain a topically acceptable diluentsuch as lactose, glucose or mannitol (preferably lactose), usually ofcomparatively large particle size e.g. an MMD of 50 μm or more, e.g. 100μm or more or a D₅₀ of 40-150 μm. As used herein, the term “lactose”refers to a lactose-containing component, including α-lactosemonohydrate, β-lactose monohydrate, α-lactose anhydrous, β-lactoseanhydrous and amorphous lactose. Lactose components may be processed bymicronization, sieving, milling, compression, agglomeration or spraydrying. Commercially available forms of lactose in various forms arealso encompassed, for example Lactohale® (inhalation grade lactose; DFEPharma), InhaLac®70 (sieved lactose for dry powder inhaler; Meggle),Pharmatose® (DFE Pharma) and Respitose® (sieved inhalation gradelactose; DFE Pharma) products. In one embodiment, the lactose componentis selected from the group consisting of α-lactose monohydrate,α-lactose anhydrous and amorphous lactose. Preferably, the lactose isα-lactose monohydrate.

Dry powder formulations may also contain other excipients such as sodiumstearate, calcium stearate or magnesium stearate.

A dry powder formulation is typically delivered using a dry powderinhaler (DPI) device. Example dry powder delivery systems includeSPINHALER, DISKHALER, TURBOHALER, DISKUS, SKYEHALER, ACCUHALER andCLICKHALER. Further examples of dry powder delivery systems includeECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER,BREEZHALER/NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN,ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIELdry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN,PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

The compounds of the invention might also be administered topically toanother internal or external surface (e.g. a mucosal surface or skin) oradministered orally. The compounds of the invention may be formulatedconventionally for such routes of administration.

The compounds of the invention are useful in the treatment of mycosesand for the prevention or treatment of disease associated with mycoses.

In an aspect of the invention there is provided use of one or morecompounds of the invention in the manufacture of a medicament for thetreatment of mycoses and for the prevention or treatment of diseaseassociated with mycoses.

In another aspect of the invention there is provided a method oftreatment of a subject with a mycosis which comprises administering tosaid subject an effective amount of one or more compounds of theinvention.

In another aspect of the invention there is provided a method ofprevention or treatment of disease associated with a mycosis in asubject which comprises administering to said subject an effectiveamount of one or more compounds of the invention.

Mycoses may, in particular, be caused by Aspergillus spp. such asAspergillus fumigatus or Aspergillus pullulans and especiallyAspergillus fumigatus. Mycoses may also be caused by Candida spp. e.g.Candida albicans or Candida glabrata, Rhizopus spp. e.g. Rhizopusoryzae, Cryptococcus spp. e.g. Cryptococcus neoformans, Chaetomium spp.e.g. Chaetomium globosum, Penicillium spp. e.g. Penicillium chrysogenumand Trichophyton spp. e.g. Trichophyton rubrum.

A disease associated with a mycosis is, for example, pulmonaryaspergillosis.

The compound of the invention may be used in a prophylactic setting byadministering the said compound prior to onset of the mycosis.

Subjects include human and animal subjects, especially human subjects.

The compounds of the invention are especially useful for the treatmentof mycoses such as Aspergillus fumigatus infection and for theprevention or treatment of disease associated with mycoses such asAspergillus fumigatus infection in at risk subjects. At risk subjectsinclude premature infants, children with congenital defects of the lungor heart, immunocompromised subjects (e.g. those suffering from HIVinfection), asthmatics, subjects with cystic fibrosis, elderly subjectsand subjects suffering from a chronic health condition affecting theheart or lung (e.g. congestive heart failure or chronic obstructivepulmonary disease).

The compounds of the invention are also useful for the treatment ofazole resistant mycoses such as azole resistant Aspergillus fumigatusinfection, particularly in combination with posaconazole.

The compounds of the invention may be administered in combination with asecond or further active ingredient. Second or further activeingredients may, for example, be selected from other anti-fungal agents(such as voriconazole or posaconazole), amphotericin B, an echinocandin(such as caspofungin) and an inhibitor of3-hydroxy-3-methyl-glutaryl-CoA reductase (such as lovastatin,pravastatin or fluvastatin).

Second or further active ingredients include active ingredients suitablefor the treatment or prevention of a mycosis such as Aspergillusfumigatus infection or disease associated with a mycosis such asAspergillus fumigatus infection or conditions co-morbid with a mycosissuch as Aspergillus fumigatus infection.

The compounds of the invention may be co-formulated with a second orfurther active ingredient or the second or further active ingredient maybe formulated to be administered separately by the same or a differentroute.

For example, the compounds of the invention may be administered topatients already being treated systemically with an anti-fungal, such asvoriconazole or posaconazole.

For example, the compounds of the invention may be co-administered e.g.co-formulated with one or more agents selected from amphotericin B, anechnocandin, such as caspofungin, and an inhibitor of3-hydroxy-3-methyl-glutaryl-CoA reductase, such as lovastatin,pravastatin or fluvastatin.

The compound of the invention may alternatively (or in addition) beco-administered e.g. co-formulated with one or more agents selected fromcandicidin, filipin, hamycin, natamycin, nystatin, rimocidin,bifonazole, butoconazole, clotrimazole, econazole, fenticonazole,isoconazole, ketoconazole, luliconazole, miconazole, omoconazole,oxiconazole. sertaconazole, sulconazole, tioconazole, albaconazole,efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole,propiconazole, ravuconazole, terconazole, abafungin, amorolfin,butenafine, naftifine, terbinafine, anidulafungin, micafungin, benzoicacid, ciclopirox, flucytosine (5-fluorocytosine), griseofulvin,tolnaftate and undecylenic acid.

Preferred combination partners include intraconazole, voriconazole,caspofungin and posaconazole.

According to an aspect of the invention there is provided a kit of partscomprising (a) a pharmaceutical composition comprising one or morecompounds of the invention optionally in combination with one or morediluents or carriers; (b) a pharmaceutical composition comprising asecond active ingredient optionally in combination with one or morediluents or carriers; (c) optionally one or more further pharmaceuticalcompositions each comprising a third or further active ingredientoptionally in combination with one or more diluents or carriers; and (d)instructions for the administration of the pharmaceutical compositionsto a subject in need thereof. The subject in need thereof may sufferfrom or be susceptible to a mycosis such as Aspergillus fumigatusinfection.

The compounds of the invention may be administered at a suitableinterval, for example once per day, twice per day, three times per dayor four times per day.

A suitable dose amount for a human of average weight (50-70 kg) isexpected to be around 50 μg to 10 mg/day e.g. 500 μg to 5 mg/dayalthough the precise dose to be administered may be determined by askilled person.

The compounds of the invention are expected to have one or more of thefollowing favourable attributes:

-   -   potent antifungal activity, particularly activity against        Aspergillus spp. such as Aspergillus fumigatus, or activity        against Candida spp., e.g. Candida albicans or Candida glabrata,        Rhizopus spp., e.g. Rhizopus oryzae, Cryptococcus spp., e.g.        Cryptococcus neoformans, Chaetomium spp., e.g. Chaetomium        globosum, Penicillium spp., e.g. Penicillium chrysogenum or        Trichophyton spp., e.g. Trichophyton rubrum, especially        following topical administration to the lung or nose;    -   a long duration of action in lungs, preferably consistent with        once daily dosing;    -   low systemic exposure following topical administration to the        lung or nose; and    -   an acceptable safety profile, especially following topical        administration to the lung or nose.

EXPERIMENTAL SECTION

Abbreviations used herein are defined below (Table 1). Any abbreviationsnot defined are intended to convey their generally accepted meaning.

TABLE 1 Abbreviations ABPA allergic bronchopulmonary aspergillosis aqaqueous ATCC American Type Culture Collection BALF bronchoalveolarlavage fluid BEAS2B SV40-immortalised human bronchial epithelial cellline Boc tert-butyloxycarbonyl br broad BSA bovine serum albumin CC₅₀50% cell cytotoxicity concentration CFU colony forming unit(s) CLSIClinical and Laboratory Standards Institute COI cut off index concconcentration/concentrated d doublet DCM dichloromethane DFB₅₀ daystaken to reach a fungal burden of 50% of control DIPEAN,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMEM Dulbecco'sModified Eagle Medium DMF N,N-dimethylformamide DMSO dimethyl sulfoxideDSS dextran sodium sulphate EBM endothelial basal media ECMextracellular matrix EDCl•HClN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride EGM2endothelial cell growth media 2 EUCAST European Committee onAntimicrobial Susceptibility Testing (ES⁺) electrospray ionization,positive mode Et ethyl Et₃N triethylamine EtOAc ethyl acetate FBS foetalbovine serum GM galactomannan HPAEC Human pulmonary artery endothelialcell HOBt•H₂O 1-hydroxybenzotriazole mono-hydrate HPLC reverse phasehigh performance liquid chromatography hr hour(s) IA invasiveaspergillosis i.n. intranasal IPA 2-propanol i.t. intra-tracheal LC-MSliquid chromatography-mass spectrometry Li Hep lithium heparin LiHMDSlithium bis(trimethylsilyl)amide m multiplet (M + H)⁺ protonatedmolecular ion MDA malondialdehyde Me methyl MeCN acetonitrile MeOHmethanol MHz megahertz MIC₅₀ 50% of minimum inhibitory concentrationMIC₇₅ 75% of minimum inhibitory concentration MIC₉₀ 90% of minimuminhibitory concentration min minute(s) MMD mass median diameter MOImultiplicity of infection MOPS 3-(N-morpholino)propanesulfonic acid m/z:mass-to-charge ratio NCPF National Collection of Pathogenic Fungi NMRnuclear magnetic resonance (spectroscopy) NT not tested OD opticaldensity PBS phosphate buffered saline P protective group a quartet RTroom temperature RP HPLC reverse phase high performance liquidchromatography RPMI Roswell Park Memorial Institute medium RuPhos2-dicyclohexylphosphino-2′, 6′-diisopropoxybiphenyl RuPhosG3(2-dicyclohexylphosphino-2′, 6′-diisopropoxybiphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium (II)methanesulfonate s singlet sat saturated scsub-cutaneous SDS sodium dodecyl sulphate t triplet TFA trifluoroaceticacid THF tetrahydrofuran TR34/L98H An Aspergillus fumigatus straincontaining a leucine-to-histidine substitution at codon 98 and a 34-bptandem repeat Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

General Procedures

All starting materials and solvents were obtained either from commercialsources or prepared according to the literature citation. Unlessotherwise stated all reactions were stirred. Organic solutions wereroutinely dried over anhydrous magnesium sulfate. Hydrogenations wereperformed on a Thales H-cube flow reactor under the conditions stated.

Column chromatography was performed on pre-packed silica (230-400 mesh,40-63 μm) cartridges using the amount indicated. SCX was purchased fromSupelco. Unless stated otherwise, the reaction mixture to be purifiedwas first diluted with DCM. This solution was loaded directly onto theSCX and washed with MeOH. The desired material was then eluted bywashing with 0.7 M NH₃ in MeOH.

Preparative Reverse Phase High Performance Liquid Chromatography

Method 1: Waters X-Select CSH column C18, 5 μm (19×50 mm), flow rate 28mL min⁻¹ eluting with a H₂O-MeCN gradient containing 0.1% v/v formicacid over 6.5 min using UV detection at 254 nm. Gradient information:0.0-0.2 min, 20% MeCN; 0.2-5.5 min, ramped from 20% MeCN to 80% MeCN;5.5-5.6 min, ramped from 80% MeCN to 95% MeCN; 5.6-6.5 min, held at 95%MeCN.

Method 2: Waters X-Select CSH column C18, 5 μm (19×50 mm), flow rate 28mL min⁻¹ eluting with a H₂O-MeCN gradient containing 0.1% v/v formicacid over 6.5 min using UV detection at 254 nm. Gradient information:0.0-0.2 min, 50% MeCN; 0.2-5.5 min, ramped from 50% MeCN to 80% MeCN;5.5-5.6 min, ramped from 80% MeCN to 95% MeCN; 5.6-6.5 min, held at 95%MeCN.

Method 3: Waters X-Select CSH column C18, 5 μm (19×50 mm), flow rate 28mL min⁻¹ eluting with a H₂O-MeCN gradient containing 0.1% v/v formicacid over 6.5 min using UV detection at 254 nm. Gradient information:0.0-0.2 min, 35% MeCN; 0.2-5.5 min, ramped from 35% MeCN to 65% MeCN;5.5-5.6 min, ramped from 65% MeCN to 95% MeCN; 5.6-6.5 min, held at 95%MeCN.

Analytical Methods

Reverse Phase HPLC Methods: Waters Xselect CSH C18 XP column, 2.5 μm(4.6×30 mm) at 40° C.; flow rate 2.5-4.5 mL min⁻¹ eluted with a H₂O-MeCNgradient containing either 0.1% v/v formic acid (Method a) or 10 mMNH₄HCO₃ in water (Method b) over 4 min employing UV detection at 254 nm.Gradient information: 0-3.00 min, ramped from 95% H₂O-5% MeCN to 5%H₂O-95% MeCN; 3.00-3.01 min, held at 5% H₂O-95% MeCN, flow rateincreased to 4.5 mL min⁻¹; 3.01 3.50 min, held at 5% H₂O-95% MeCN;3.50-3.60 min, returned to 95% H₂O-5% MeCN, flow rate reduced to 3.50 mLmin⁻¹; 3.60-3.90 min, held at 95% H₂O-5% MeCN; 3.90-4.00 min, held at95% H₂O-5% MeCN, flow rate reduced to 2.5 mL min⁻¹.

¹H NMR Spectroscopy: ¹H NMR spectra were acquired on a Bruker AdvanceIII spectrometer at 400 MHz using residual undeuterated solvent asreference and unless specified otherwise were run in DMSO-d₆.

Representative Procedures for the Preparation of Intermediates

tert-butyl 4-(4-hydroxy-3-methylphenyl)piperazine-1-carboxylate

A flask charged with tert-butylpiperazin-1-carboxylate (7.44 g, 40.0mmol), 4-bromo-2-methylphenol (Xa) (6.23 g, 33.3 mmol), RuPhos (311 mg,0.67 mmol) and RuPhos G3 (557 mg, 0.67 mmol) was evacuated andbackfilled with nitrogen three times. A solution of LiHMDS (1M in THF,100 mL, 100 mmol) was added and the reaction mixture was heated at 70°C. for 3 hr. After cooling to RT the mixture was quenched by theaddition of 1M hydrochloric acid (100 mL) and was then neutralised with1M aq. NaHCO₃ (100 mL). The aq layer was extracted with EtOAc (3×100 mL)and the combined organic extracts were dried. The volatiles were removedin vacuo to give a crude product which was purified by flash columnchromatography (SiO₂, 120 g, 0-100% EtOAc in isohexanes, gradientelution) to afford the title compound, intermediate (XIIa), as a lightbrown solid (7.80 g, 78%); R^(t) 2.07 min (Method b); m/z 293 (M+H)⁺(ES⁺); ¹H NMR δ: 1.41 (9H, s), 2.07 (3H, s), 2.86-2.88 (4H, m),3.41-3.43 (4H, m), 6.58-6.65 (2H, m), 6.71 (1H, d) and 8.72 (1H, s).

1-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazine

To a solution of intermediate (XIIa) (7.80 g, 25.1 mmol) in DMSO (60 mL)was added aq sodium hydroxide (3.0 mL, 12.5 M, 37.6 mmol). The mixturewas stirred at RT for 10 min and was then treated portionwise with thetosylate (XIa) (ex APIChem, Catalogue Number: AC-8330, 12.4 g, 27.6mmol). The reaction mixture was stirred at 30° C. for 18 hr, cooled toRT and water (200 mL) was added. The resulting mixture was extractedwith EtOAc (3×200 mL) and the combined organic extracts were washed withbrine (2×200 mL), and then dried and evaporated in vacuo to afford abrown oil. Analysis of the crude, Boc-protected product (VIIa) by ¹H NMRindicated that it contained ˜10% of the alkene:(R)-1-((2-(2,4-difluorophenyl)-4-methylenetetrahydrofuran-2-yl)methyl)-1H-1,2,4-triazole,formed as an elimination by-product. The crude urethane (VIIa) was takenup into DCM (150 mL) and treated with TFA (39.0 mL, 502 mmol). After 2hr at RT the reaction mixture was concentrated in vacuo to remove mostof the volatiles and was then diluted with EtOAc (200 mL) and washedwith aq. NaOH (2M, 200 mL). The aq phase was separated and was extractedwith EtOAc (2×200 mL). The combined organic extracts were washed withbrine (2×200 mL) and then dried and evaporated in vacuo to afford alight brown oil. The crude product was purified by flash columnchromatography (SiO₂, 80 g, 0-10% 0.7 M NH₃/MeOH in DCM, gradientelution) to afford the title compound, intermediate (Va), as a viscous,light brown oil (9.46 g, 80%); R^(t) 1.91 min (Method b); m/z 470 (M+H)⁺(ES⁺); ¹H NMR δ: 2.07 (3H, s), 2.15 (1H, dd), 2.36-2.42 (1H, m),2.52-2.56 (1H, m), 2.79-2.81 (4H, m), 2.87-2.90 (4H, m), 3.66 (1H, dd),3.73-3.77 (2H, m), 4.04 (1H, t), 4.57 (2H, dd), 6.64 (1H, dd), 6.70-6.75(2H, m), 6.99 (1H, td), 7.25-7.34 (2H, m), 7.76 (1H, s) and 8.34 (1H,s).

Methyl4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzoate

A flask charged with intermediate (Va) (9.00 g, 19.2 mmol),methyl-4-bromobenzoate (VIa) (4.95 g, 23.0 mmol), RuPhos (0.18 g, 0.38mmol, 2 mol %), RuPhosG3 (0.32 g, 0.38 mmol, 2 mol %) and cesiumcarbonate (9.99 g, 30.7 mmol) was evacuated and refilled with nitrogenthree times before DMF (150 mL) was added. The mixture was heated at 80°C. for 22 hr and then, whilst still hot, was poured into water (150 mL)to form a brown gum. More water (300 mL) was added and the aq. phase wasextracted with DCM (2×200 mL). The organic extracts were combined andconcentrated in vacuo to give a brown oil which was poured into water(100 mL). The resulting precipitate was collected by filtration and thenre-suspended in THF (100 mL). The mixture was heated at reflux for 1 hrduring which time a cream suspension was formed. The mixture was cooledto RT and the resulting precipitate was collected by filtration, washedwith THF (2×50 mL) and then dried in vacuo to afford the title compound,intermediate (IVa), as a light yellow solid (9.48 g, 79%); R^(t) 2.79min (Method b); m/z 604 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.09 (3H, s), 2.16 (1H,dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.11-3.14 (4H, m), 3.43-3.46(4H, m), 3.68 (1H, dd), 3.74-3.79 (5H, s overlapping over m), 4.05 (1H,dd), 4.58 (2H, dd), 6.75 (2H, br s), 6.85 (1H, br d), 7.00 (1H, td),7.04 (2H, d), 7.25-7.34 (2H, m), 7.76 (1H, s), 7.81 (2H, d) and 8.34(1H, s).

Ethyl 4-(4-(4-hydroxy-3-methylphenyl)piperazin-1-yl)benzoate

A flask charged with a solution of ethyl 4-(piperazin-1-yl)benzoate(XIVa) (20.0 g, 85.0 mmol) and 4-bromo-2-methylphenol (Xa) (19.2 g, 102mmol) in DMF (213 mL) was evacuated and backfilled with nitrogen threetimes. RuPhos G3 (1.43 g, 1.71 mmol) was added and the flask wasevacuated and backfilled with nitrogen. The reaction mixture was cooledto 0° C. and LiHMDS (17.1 g, 102 mmol) was added. The reaction wasstirred at RT for 10 min, then cooled in a water bath and LiHMDS (20.0g, 120 mmol) added in equal portions (7×2.85 g) at 5 min intervals. Theresulting solution was stirred at RT for 30 min and was then cooled to0° C. and treated with 2M hydrochloric acid (200 mL) resulting in a pHof 6-7. The mixture was stirred for 15 min at RT and was then extractedwith EtOAc (220 mL). The aq layer was separated and extracted with EtOAc(4×50 mL) and the combined organics were washed with brine (6×50 mL),and then dried and evaporated in vacuo to afford a cream solid. Amixture of isohexanes and IPA (1:1, 150 mL) was added and the suspensionwas stirred at RT for 30 min. The solid was collected by filtration, andthe filter cake was washed with a mixture of isohexanes and IPA (1:1,2×10 mL) followed by isohexanes (4×10 mL) and dried in vacuo at 40° C.for 18 hr to afford the title compound, intermediate (XIIIa), as a creamsolid (15.3 g, 50%); R^(t) 2.29 min (Method b); m/z 341 (M+H)⁺ (ES⁺); ¹HNMR δ: 1.29 (3H, t), 2.09 (3H, s), 3.06-3.09 (4H, m), 3.42-3.44 (4H, m),4.24 (2H, dd), 6.66 (2H, br s), 6.76 (1H, br s), 7.03 (2H, d), 7.80 (2H,d), 8.72 (1H, 5).

Ethyl4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzoate

To a solution of intermediate (XIIIa) (15.3 g, 44.9 mmol) in DMF (110mL) cooled to 0° C. was added sodium ethoxide (3.13 g, 46.1 mmol) andthe mixture stirred at 0° C. for 10 min and then treated with thetosylate (XIa). The reaction mixture was allowed to warm to RT, heatedto 50° C. for 1 hr and then cooled to RT. Hydrochloric acid (1M, 60 mL)and water (200 mL) were added and the mixture was stirred for 30 min atRT and then extracted with DCM (150 mL). The aq layer was separated andextracted with DCM (2×50 mL) and the combined organics were washed withbrine (4×30 mL) and then dried and evaporated in vacuo to afford a creamsolid. The solid was suspended in an equal mixture of isohexanes and IPA(80 mL) and stirred at RT for 1 hr. The solid was collected byfiltration, washed with a mixture of isohexanes and IPA 1:1 (3×20 mL)and then dried in vacuo at 40° C. for 18 hr to afford the titlecompound, intermediate (IVb) as a white solid (16.4 g, 56%); R^(t) 2.92min (Method b); m/z 618 (M+H)⁺ (ES⁺); ¹H NMR δ: 1.29 (3H, t), 2.10 (3H,s), 2.16 (1H, dd), 2.37-2.42 (1H, m), 2.52-2.58 (1H, m), 3.12-3.14 (4H,m), 3.43-3.46(4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd),4.24 (2H, dd), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s),6.98-7.05 (3H, m), 7.26-7.34 (2H, m), 7.77 (1H, s), 7.81 (2H, d), 8.34(1H, s).

2-(Benzyloxy)-5-bromobenzonitrile

To a stirred suspension of 5-bromo-2-hydroxybenzonitrile (Xb) (1.00 g,5.05 mmol), tetrabutylammonium bromide (814 mg, 2.53 mmol) and potassiumphosphate tribasic monohydrate (1.16 g, 5.05 mmol) in water (10 mL) wasadded benzyl bromide (600 μL, 5.05 mmol). The mixture was stirred for 18hr at RT and was extracted with DCM (3×20 mL). The combined organicswere washed with brine (20 mL) and then dried and evaporated in vacuo.The crude product so obtained was purified by flash columnchromatography (SiO₂, 12 g, 0-40% EtOAc in isohexanes, gradient elution)to afford the title compound, intermediate (Xc), as a white solid (1.22g, 82%); R^(t) 2.53 min (Method a); m/z no ionisation observed; ¹H NMRδ: 5.30 (2H, s), 7.31 (1H, d), 7.34-7.48 (5H, m), 7.85 (1H, dd), 8.03(1H, d).

Methyl 4-(4-(4-(benzyloxy)-3-cyanophenyl)piperazin-1-yl)benzoate

A flask charged with methyl 4-(piperazin-1-yl)benzoate (XIVa) (459 mg,2.08 mmol), intermediate (Xc) (500 mg, 1.74 mmol), RuPhos (40.0 mg, 87.0μmol), RuPhos G3 (67.0 mg, 87.0 μmol) and cesium carbonate (678 mg, 2.08mmol) was evacuated and backfilled with nitrogen three times before DMF(8.0 mL) was added. The mixture was heated at 80° C. for 18 hr and wasthen cooled to RT and partitioned between water (50 mL) and EtOAc (50mL). The organic phase was separated and retained and the aq layer wasextracted with EtOAc (2×50 mL). The combined organics were washed withbrine (3×50 mL) and then dried and evaporated in vacuo to afford ayellow solid. The crude product was triturated in diethyl ether (20 mL),collected by filtration and dried in vacuo at 40° C. for 18 hr to givethe title compound, (XIIIb) as a beige solid (286 mg, 37%); R^(t) 2.74min (Method a); m/z 428 (M+H)⁺ (ES⁺); ¹H NMR δ: 3.21-3.24 (4H, m),3.44-3.47 (4H, m), 3.78 (3H, s), 5.22 (2H, s), 7.05 (2H, d), 7.22-7.25(1H, m), 7.32-7.36 (3H, m), 7.39-7.47 (4H, m), 7.81 (2H, d).

Methyl 4-(4-(3-cyano-4-hydroxyphenyl)piperazin-1-yl)benzoate

A solution of intermediate (XIIIb) (286 mg, 0.669 mmol) in EtOAc (80 mL)was hydrogenated using the H-Cube (10% palladium on carbon, 70×4 mm,full hydrogen, 30° C., 1 mL/min). The solvent was evaporated in vacuoand the residue was purified by flash column chromatography (SiO₂, 4 g,0-100% EtOAc in DCM, gradient elution) to furnish the title compound,intermediate (XIIIc), as a white solid (139 mg, 60%); R^(t) 2.06 min(Method a); m/z 338 (M+H)⁺ (ES⁺); ¹H NMR δ: 3.14-3.17 (4H, m), 3.43-3.46(4H, m), 3.78 (3H, s), 6.93 (1H, d), 7.04 (2H, d), 7.16 (1H, d), 7.25(1H, dd), 7.80 (2H, d), 10.45 (1H, s).

Methyl4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-cyanophenyl)piperazin-1-yl)benzoate

To a solution of intermediate (XIIIc) (139 mg, 0.412 mmol) in DMSO (1.0mL) was added aq sodium hydroxide (30.0 μL, 12.5 M, 0.381 mmol) and themixture stirred at RT for 10 min and then treated with a solution of thetosylate (XIa) (154 mg, 0.343 mmol) in DMSO (1.0 mL). The reactionmixture was stirred at 30° C. for 18 hr, cooled to RT and water (30 mL)was added. The resulting mixture was extracted with EtOAc (3×50 mL) andthe combined organic extracts were washed with brine (2×30 mL) and driedand evaporated in vacuo to give a brown oil. The crude product waspurified by flash column chromatography (SiO₂, 12 g, 0-100% EtOAc inDCM, gradient elution) to give the title compound, intermediate (IVc),as a white foam (100 mg, 46%); R^(t) 2.59 min (Method a); m/z 615 (M+H)⁺(ES⁺); ¹H NMR δ: 2.18 (1H, dd), 2.40-2.46 (1H, m), 2.56-2.64 (1H, m),3.21-3.24 (4H, m), 3.45-3.47 (4H, m), 3.78-3.96 (6H, m), 4.04 (1H, dd),4.59 (2H, dd), 6.98 (1H, td), 7.05 (2H, d), 7.11 (1H, d), 7.25-7.35 (4H,m), 7.73 (1H, s), 7.81 (2H, d), 8.33 (1H, s).

1-(((2R,4R)-4-((4-bromo-2-methylphenoxy)methyl)-2-(2,4-difluorophenyl)tetrahydrofuran-2-yl)methyl)-1H-1,2,4-triazole

To a solution of 4-bromo-2-methyl phenol (Xa) (920 mg, 4.89 mmol) inDMSO (10 mL) was added aq sodium hydroxide (0.39 mL, 12.5 M, 4.89 mmol)and the mixture stirred at RT for 10 min and then treated with thetosylate (XIa) (2.00 g, 4.45 mmol). The reaction mixture was stirred at60° C. for 72 hr then cooled to RT and partitioned between water (25 mL)and EtOAc (20 mL). The organic phase was separated and retained and theaq layer was extracted with EtOAc (3×25 mL). The combined organicextracts were washed with brine (3×15 mL) and then dried and evaporatedin vacuo. The crude product was purified by flash column chromatography(SiO₂, 12 g, 0-30% EtOAc in DCM, gradient elution) to give the titlecompound, intermediate (VIIIa), as a colourless oil (1.84 g, 86%); R^(t)2.78 min (Method a); m/z 464 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.09 (3H, s), 2.17(1H, dd), 2.37-2.43 (1H, m), 2.52-2.60 (1H, m), 3.72-3.78 (2H, m), 3.82(1H, dd), 4.00-4.06 (1H, m), 4.57 (2H, dd), 6.82 (1H, d), 7.00 (1H, td),7.25-7.34 (4H, m), 7.76 (1H, s), 8.34 (1H, s).

Ethyl4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzoate

A vial charged with ethyl 4-(piperazin-1-yl)benzoate (XIVa) (103 mg,0.44 mmol), intermediate (VIIIa) (170 mg, 0.37 mmol), RuPhos (8.5 mg, 18μmol), RuPhos G3 (14.2 mg, 18 μmol) and cesium carbonate (191 mg, 0.59mmol) was evacuated and backfilled with nitrogen three times before DMF(3.0 mL) was added. The mixture was heated at 80° C. for 18 h and thenat 100° C. for 24 hr. The reaction mixture was cooled to RT andpartitioned between water (10 mL) and EtOAc (10 mL). The organic phasewas separated and retained and the aq layer was extracted with EtOAc(3×10 mL). The combined organics were washed with brine (3×10 mL) andthen dried and evaporated in vacuo. The crude product was purified byflash column chromatography (SiO₂, 12 g, 0-100% EtOAc in isohexane,gradient elution) to give the title compound, intermediate (IVb), as awhite solid (100 mg, 43%).

3-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-bromo-2-methylpyridine

To a solution of 6-bromo-2-methylpyridin-3-ol (Xd) (1.00 g, 5.32 mmol)in DMSO (17 mL) was added aq sodium hydroxide (2.80 mL, 2.0 M, 5.32mmol) and the mixture stirred at RT for 10 min and then treatedportionwise with the tosylate (XIa) (2.17 g, 4.84 mmol). The reactionmixture was stirred at 65° C. for 18 hr, cooled to RT and water (30 mL)was added. The resulting mixture was extracted with EtOAc (3×50 mL) andthe combined organics were washed with brine (2×30 mL) and then driedand evaporated in vacuo to afford a yellow oil. The crude product waspurified by flash column chromatography (SiO₂, 40 g, 0-100% EtOAc inDCM, gradient elution) to afford the title compound, intermediate(VIIIb), as a white solid (1.50 g, 64%); R^(t) 2.28 min (Method a); m/z465/467 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.17 (1H, dd), 2.29 (3H, s), 2.38-2.44(1H, m), 2.54-2.62 (1H, m), 3.74-3.88 (3H, m), 4.03 (1H, dd), 4.58 (2H,dd), 7.00 (1H, td), 7.25-7.35 (3H, m), 7.39 (1H, dd), 7.76 (1H, s), 8.34(1H, s).

tert-Butyl4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)piperazine-1-carboxylate

A flask charged with tert-butylpiperazin-1-carboxylate (IXa) (550 mg,2.93 mmol), intermediate (VIIIb) (1.50 g, 3.22 mmol), RuPhos (68.0 mg,0.147 mmol), RuPhos G3 (123 mg, 0.147 mmol) and cesium carbonate (1.53g, 4.69 mmol) was evacuated and backfilled with nitrogen three timesbefore DMF (15 mL) was added. The mixture was heated at 80° C. for 18hr, then cooled to RT and partitioned between water (100 mL) and EtOAc(100 mL). The organic phase was separated and retained and the aq layerwas extracted with EtOAc (2×100 mL). The combined organic extracts werewashed with brine (3×50 mL) and then dried and evaporated in vacuo toafford a yellow oil. The crude product was purified by flash columnchromatography (SiO₂, 80 g, 0-100% EtOAc in DCM, gradient elution) toafford the title compound, intermediate (VIIb), as a white foam (1.38 g,77%); R^(t) 2.14 min (Method a); m/z 571 (M+H)⁺ (ES⁺); ¹H NMR δ: 1.41(9H, s), 2.15 (1H, dd), 2.21 (3H, s), 2.36-2.42 (1H, m), 2.52-2.57 (1H,m), 3.29-3.33 (4H, m), 3.39-3.41 (4H, m), 3.67 (1H, dd), 3.75 (2H, dd),4.04 (1H, dd), 4.57 (2H, dd), 6.60 (1H, d), 7.00 (1H, td), 7.18 (1H, d),7.25-7.34 (2H, m), 7.76 (1H, s), 8.34 (1H, s).

1-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)piperazine

A solution of intermediate (VIIb) in DCM (10 mL) was treated with TFA(2.62 mL, 34.0 mmol) and stirred at RT for 2 hr. The reaction mixturewas concentrated in vacuo to give a brown oil, which was partitionedbetween EtOAc (50 mL) and 1M aq NaHCO₃ (20 mL). The organic layer wasseparated and was washed with brine (20 mL) and then dried andevaporated in vacuo to afford the title compound, intermediate (Vb) as abrown foam (858 mg, 76%); R^(t) 1.26 min (Method a); m/z 471 (M+H)⁺(ES⁺); ¹H NMR δ: 2.14 (1H, dd), 2.21 (3H, s), 2.36-2.42 (1H, m),2.53-2.57 (1H, m), 2.80-2.85 (4H, m), 3.26-3.31 (4H, m), 3.66 (1H, dd),3.71-3.77 (2H, dd), 4.04 (1H, t), 4.57 (2H, dd), 6.56 (1H, d), 7.00 (1H,td), 7.17 (1H, d), 7.25-7.34 (2H, m), 7.76 (1H, s), 8.34 (1H, s).

Methyl4-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)piperazin-1-yl)benzoate

A flask charged with intermediate (Vb) (860 mg, 1.82 mmol),methyl-4-bromobenzoate (VIa) (470 mg, 2.19 mmol), RuPhos (17.0 mg,0.0360 mmol, 2 mol %), RuPhosG3 (28.0 mg, 0.0360 mmol, 2 mol %) andcesium carbonate (950 mg, 2.92 mmol) was evacuated and refilled withnitrogen three times before DMF (6.0 mL) was added. The mixture washeated at 80° C. for 18 hr, then cooled to RT and partitioned betweenEtOAc (50 mL) and water (50 mL). The organic layer was separated and waswashed with brine (3×50 mL) and then dried and evaporated in vacuo togive a beige solid. The crude product so obtained was purified by flashcolumn chromatography (SiO₂, 24 g, 0-100% EtOAc in DCM, gradientelution) to afford the title compound, intermediate (IVd), as a yellowsolid (690 mg, 61%); R^(t) 2.21 min (Method a); m/z 605 (M+H)⁺ (ES⁺); ¹HNMR δ: 2.15 (1H, dd), 2.23 (3H, s), 2.37-2.43 (1H, m), 2.52-2.57 (1H,m), 3.42-3.44 (4H, m), 3.47-3.50 (4H, m), 3.68 (1H, dd), 3.74-3.78 (5H,m), 4.04 (1H, dd), 4.57 (2H, dd), 6.66 (1H, d), 6.97-7.04 (3H, td), 7.21(1H, d), 7.25-7.34 (2H, m), 7.76 (1H, s), 7.81 (2H, d), 8.34 (1H, s).

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzoicacid

Hydrolysis of the Methyl Ester (IVa)

To a suspension of intermediate (IVa) (9.00 g, 14.9 mmol) in DMSO (370mL) was added a solution of lithium hydroxide (1.79 g, 74.5 mmol) inwater (37.0 mL). The mixture was heated at 70° C. for 22 hr and was thencooled to RT, diluted with water (1000 mL) and acidified (to ˜ pH 2) bythe addition of 1M aq hydrochloric acid (80 mL). The mixture was cooledin an ice bath for 2 hr and the resulting precipitate was collected byfiltration. The filter cake was washed with water (3×80 mL) and dried invacuo at 50° C. to give the title compound, intermediate (IIa) as awhite solid (4.66 g, 54%); R^(t) 2.21 min (Method 1a); m/z 590 (M+H)⁺(ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m),2.52-2.58 (1H, m), 3.12-3.14 (4H, m), 3.42-3.45 (4H, m), 3.68 (1H, dd),3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58 (2H, dd), 6.76 (2H, br s), 6.86(1H, br d), 6.97-7.03 (3H, m), 7.25-7.34 (2H, m), 7.77-7.80 (3H, m),8.34 (1H, s) and 12.31 (1H, s).

Hydrolysis of the Ethyl Ester (IVb)

To a suspension of intermediate (IVb) (16.4 g, 26.6 mmol) in DMSO (375mL) was added a solution of lithium hydroxide (3.18 g, 74.5 mmol) inwater (50 mL). The mixture was heated at 70° C. for 22 hr and was thencooled to RT, poured into water (500 mL) and acidified (to ˜ pH 5-6) bythe addition of 2M hydrochloric acid (70 mL). The mixture was stirred atRT for 30 min and the resulting solid was collected by filtration andwashed with water (2×20 mL) and with diethyl ether (3×30 mL) and thendried in vacuo at 40° C. for 18 hr to afford the title compound,intermediate (IIa) as a tan solid (14.2 g, 84%); R^(t) 2.26 min (Method1a); m/z 590 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.09 (3H, s), 2.16 (1H, dd),2.37-2.42 (1H, m), 2.52-2.58 (1H, m), 3.12-3.14 (4H, m), 3.42-3.44 (4H,m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58 (2H, dd), 6.75(2H, br s), 6.86 (1H, br s), 6.97-7.03 (3H, m), 7.26-7.34 (2H, m),7.77-7.80 (3H, m), 8.34 (1H, s), 12.31 (1H, br s).

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-cyanophenyl)piperazin-1-yl)benzoicacid

To a suspension of intermediate (IVc) (100 mg, 0.163 mmol) in DMSO (8.0mL) was added a solution of lithium hydroxide (19 mg, 0.81 mmol) inwater (1.0 mL). The mixture was heated at 50° C. for 18 hr, then cooledto RT, diluted with water (10 mL) and acidified (to ˜ pH 3) by theaddition of 1M hydrochloric acid (2.0 mL). The mixture was extractedwith 4:1 DCM/EtOAc (2×25 mL) and the combined organics were washed withwater (2×10 mL) and dried and evaporated in vacuo to afford a whitesolid that was dried in vacuo at 40° C. to give the title compound,intermediate (IIb) as a white solid (74 mg, 75%); R^(t) 2.32 min (Methoda); m/z 601 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.18 (1H, dd), 2.40-2.46 (1H, m),2.58-2.64 (1H, m), 3.21-3.24 (4H, m), 3.43-3.45 (4H, m), 3.78-3.96 (3H,m), 4.04 (1H, dd), 4.59 (2H, dd), 6.98 (1H, td), 7.03 (2H, d), 7.11 (1H,d), 7.25-7.35 (4H, m), 7.74 (1H, s), 7.79 (2H, d), 8.34 (1H, s), 12.30(1H, br s).

4-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)piperazin-1-yl)benzoicacid

To a suspension of intermediate (IVd) (690 mg, 1.14 mmol) in DMSO (54mL) was added a solution of lithium hydroxide (140 mg, 5.71 mmol) inwater (9.0 mL). The mixture was heated at 70° C. for 22 hr and thencooled to RT, diluted with water (100 mL) and acidified (to ˜ pH 2) bythe addition of 1M hydrochloric acid (6.0 mL). The mixture was cooled inan ice bath for 15 min and the resulting precipitate was collected byfiltration, washed with water (3×50 mL) and dried in vacuo at 40° C. togive the title compound, intermediate (IIc), as a yellow solid (617 mg,87%); R^(t) 1.91 min (Method a); m/z 591 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.15(1H, dd), 2.23 (3H, s), 2.37-2.43 (1H, m), 2.52-2.57 (1H, m), 3.39-3.42(4H, m), 3.47-3.50 (4H, m), 3.68 (1H, dd), 3.74-3.78 (2H, m), 4.04 (1H,dd), 4.57 (2H, dd), 6.66 (1H, d), 6.97-7.02 (3H, td), 7.21 (1H, d),7.25-7.34 (2H, m), 7.76-7.80 (3H, m), 8.34 (1H, s), 12.31 (1H, br s).

4-Bromo-N-(4-fluorophenyl)benzamide

To a solution of 4-fluoroaniline (IIIa) (0.85 mL, 9.00 mmol),triethylamine (1.88 mL, 13.5 mmol) and DMAP (0.11 g, 0.90 mmol) in THF(15 mL) was added 4-bromobenzoyl chloride (XIXa) (2.37 g, 10.8 mmol).The reaction mixture was maintained at RT for 1 hr and was thenpartitioned between EtOAc (100 mL) and 1M hydrochloric acid (100 mL).The organic phase was separated and was washed sequentially with 1Mhydrochloric acid (100 mL), sat. aq. NaHCO₃ (100 mL) and brine (100 mL)and then dried and evaporated in vacuo. The crude residue was trituratedfrom warm DCM (100 mL) and the mixture was heated at reflux to give awhite suspension which was allowed to cool to RT. The resultingprecipitate was collected by filtration to afford the title compound,intermediate (XVIIIa), as white solid (1.81 g, 65%); R^(t) 2.23 min; m/z294/296 (M+H)⁺ (ES⁺); ¹H NMR δ: 7.20 (2H, t), 7.74-7.79 (4H, m), 7.90(2H, d) and 10.36 (1H, s).

tert-Butyl4-(4-((4-fluorophenyl)carbamoyl)phenyl)piperazine-1-carboxylate

A flask charged with tert-butyl piperazine-1-carboxylate (IXa) (4.00 g,215 mmol), intermediate (XVIIIa) (6.63 g, 22.6 mmol), RuPhos (100 mg,0.215 mmol) and RuPhos G3 (180 mg, 0.215 mmol) was evacuated andbackfilled with nitrogen three times. A solution of LiHMDS (1M in THF,75.0 mL, 75.0 mmol) was added and the reaction mixture was heated at 70°C. for 5 hr. After cooling to RT the mixture was partitioned betweenEtOAc (150 mL) and 1M hydrochloric acid (150 mL). The organic phase wasseparated and retained and the aq phase was extracted with EtOAc (3×150mL). The combined organics were dried and concentrated in vacuo toafford a brown solid which was triturated in a mixture of isohexanes anddiethyl ether (1:1, 100 mL). The product so obtained was collected byfiltration, washed with a mixture of isohexanes and diethyl ether (1:1,25 mL) and then dried in vacuo at 40° C. to provide the title compound,intermediate (XVIIa) as a tan solid (6.44 g, 85%); R^(t) 2.40 min(Method a); m/z 400 (M+H)⁺; ¹H NMR δ: 1.43 (9H, s), 3.27-3.30 (4H, m),3.45-3.48 (4H, m), 7.03 (2H, d), 7.14-7.18 (2H, m), 7.74-7.79 (2H, m),7.88 (2H, d), 9.99 (1H, s).

N-(4-fluorophenyl)-4-(piperazin-1-yl)benzamide

To a solution of intermediate (XVIIa) (6.44 g, 16.1 mmol) in DCM (200mL) was added TFA (24.7 mL, 322 mmol). The reaction was stirred at RTfor 2 hr and was then evaporated in vacuo. Toluene (5.0 mL) was addedand the mixture was again evaporated in vacuo. The resulting oil wastaken up in a mixture of DCM (90 mL) and methanol (10 mL) and was thenextracted with a mixture of water (50 mL) and sat. aq NaHCO₃ (50 mL).The organic phase was separated and retained and the aq layer wasextracted with a mixture of DCM and methanol (9:1, 3×100 mL). Thecombined organic layers were dried and concentrated in vacuo to affordthe title compound, intermediate (XVIa), as a brown solid (3.74 g, 70%);R^(t) 1.02 min (Method a); m/z 300 (M+H)⁺; ¹H NMR δ: 2.81-2.83 (4H, m),3.18-3.20 (4H, m), 6.99 (2H, d), 7.14-7.18 (2H, m), 7.74-7.80 (2H, m),7.85 (2H, d), 9.99 (1H, s).

N-(4-fluorophenyl)-4-(4-(4-methoxy-3-methylphenyl)piperazin-1-yl)benzamide

A flask charged with 4-bromo-1-methoxy-2-methylbenzene (Xe) (406 mg,2.02 mmol), intermediate (XVIa) (550 mg, 1.84 mmol), RuPhos (43 mg,0.092 mmol) and RuPhos G3 (77 mg, 0.092 mmol) was evacuated andbackfilled with nitrogen three times. A solution of LiHMDS (9.2 mL, 1Min THF, 9.2 mmol) was added and the reaction mixture was heated at 70°C. for 8 hr. After cooling to RT the mixture was quenched by theaddition of 1M aq. hydrochloric acid (9.0 mL) and then partitionedbetween water (15 mL) and EtOAc (15 mL). The organic layer was separatedand retained and the aq layer was extracted with EtOAc (2×15 mL). Thecombined organics were washed with brine (20 mL) and then dried andevaporated in vacuo. The crude product so obtained was purified by flashcolumn chromatography (SiO₂, 12 g, 0-100% EtOAc in isohexane, gradientelution) to afford a yellow solid. This material was repurified by flashcolumn chromatography (SiO₂, 4 g, 0-10% EtOAc in DCM, gradient elution)to afford the title compound, intermediate (XVa), as an off-white solid(83 mg, 11%); R^(t) 2.27 min (Method a); m/z 420 (M+H)⁺ (ES⁺); ¹H NMR δ:2.13 (3H, s), 3.13-3.16 (4H, m), 3.42-3.45 (4H, m), 3.72 (3H, s),6.77-6.88 (3H, m), 7.08 (2H, d), 7.17 (2H, t), 7.75-7.80 (2H, m), 7.89(2H, d), 10.02 (1H, s).

N-(4-fluorophenyl)-4-(4-(4-hydroxy-3-methylphenyl)piperazin-1-yl)benzamide

To a suspension of intermediate (XVa) (83 mg, 0.20 mmol) in DCM (5.0 mL)at 0° C. was added a solution of boron tribromide (0.59 mL, 1M in DCM,0.59 mmol). The reaction mixture was stirred at 0° C. for 30 min,allowed to warm to RT for 8 hr and was then partitioned between water(15 mL) and DCM (10 mL). The organic layer was separated and retainedand the aq layer was extracted with a mixture of DCM and MeOH (90:10,5×15 mL). The combined organics were dried and evaporated in vacuo togive a crude product which was purified by flash column chromatography(SiO₂, 4.0 g, 0-3% MeOH in DCM, gradient elution) to afford the titlecompound, intermediate (XVb), as a beige solid (61 mg, 72%); R^(t) 1.73min (Method a); m/z 406 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 3.08-3.11(4H, m), 3.41-3.43 (4H, m), 6.67 (2H, br s), 6.77 (1H, br s), 7.07 (2H,d), 7.17 (2H, t), 7.76-7.80 (2H, m), 7.89 (2H, d), 8.73 (1H, s), 10.01(1H, s).

N-(4-fluorophenyl)-4-(4-(4-hydroxy-2,5-dimethylphenyl)piperazin-1-yl)benzamide

A flask charged with 4-bromo-2,5-dimethylphenol (Xf) (73.1 mg, 0.364mmol), intermediate (XVIa) (120 mg, 0.400 mmol), RuPhos (8.48 mg, 0.0180mmol) and RuPhos G3 (15.2 mg, 0.0180 mmol) was evacuated and backfilledwith nitrogen three times. A solution of LiHMDS (1M in THF, 1.46 mL,1.46 mmol) was added and the reaction mixture was heated at 70° C. for18 hr. After cooling to RT the mixture was quenched by the addition of 1M hydrochloric acid (5.0 mL) and was then basified with 2M aq. NaOH (10mL). The aq layer was extracted with EtOAc (3×15 mL) and the combinedorganics dried and evaporated in vacuo. The crude product so obtainedwas purified by flash column chromatography (SiO₂, 24 g, 0-5% methanol(1% NH₃) in DCM, gradient elution) to afford a brown solid. The solidwas triturated in diethyl ether (20 mL) and collected by filtration,washed with diethyl ether (10 mL) and dried at 40° C. in vacuo to affordthe title compound, intermediate (XVc), as an off-white solid (72.0 mg,47%); R^(t) 2.19 min (Method a); m/z 420 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.07(3H, s), 2.18 (3H, s), 2.87-2.89 (4H, m), 3.39-3.42 (4H, m), 6.60 (1H,s), 6.80 (1H, s), 7.06 (2H, d), 7.14-7.19 (2H, m), 7.76-7.80 (2H, m),7.89 (2H, d), 8.81 (1H, s), 10.00 (1H, s).

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)benzamide

To a solution of intermediate (IIa) (1.00 g, 1.70 mmol), DIPEA (1.78 mL,10.2 mmol) and ammonium chloride (0.454 g, 8.48 mmol) in DMF (30 mL) at0° C. was added HATU (1.30 g, 3.39 mmol) portionwise over 2 min. Thereaction mixture was warmed to RT for 2 hr, then diluted with water (70mL) and the resulting solid collected by filtration. The filter cake waswashed with water (2×50 mL) and the solid dried in vacuo at 40° C. togive the title compound, intermediate (XXa) as an off-white powder (850mg, 83%); R^(t) 2.24 min (Method b); m/z 589 (M+H)⁺ (ES⁺); ¹H NMR δ:2.09 (3H, s), 2.16 (1H, dd), 2.37-2.42 (1H, m), 2.51-2.57 (1H, m),3.11-3.14 (4H, m), 3.36-3.39 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m),4.04 (1H, t), 4.58 (2H, dd), 6.75 (2H, br s), 6.85 (1H, br s), 6.97-7.04(4H, br s), 7.26-7.34 (2H, m),7.72 (1H, br s), 7.77 (3H, t), 8.34 (1H,s).

Preparation of Compound Examples of the Invention EXAMPLE 14-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide

1. From Intermediate (IIa)

To a suspension of intermediate (IIa) (2.50 g, 4.24 mmol), EDCI (1.63 g,8.48 mmol) and DMAP (30 mg, 0.21 mmol) in pyridine (30 mL) was added4-fluoroaniline (0.41 mL, 4.3 mmol) and the reaction mixture heated at60° C. for 2 hr and then cooled to RT. Dilution of the mixture withwater (60 mL) and stirring for 5 min produced a solid, which wascollected by filtration and then washed with water (3×10 mL) and withdiethyl ether (2×15 mL) to give a tan coloured powder. The crude productso obtained was purified by flash column chromatography (SiO₂, 40 g,0-3% MeOH in DCM, gradient elution) to afford the title compound,Example 1, as a yellow solid (2.47 g, 85%); R^(t) 2.60 min (Method a);m/z 683 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.15 (1H, dd), 2.37-2.43(1H, m), 2.53-2.58 (1H, m), 3.13-3.16 (4H, m), 3.42-3.44 (4H, m), 3.68(1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58 (2H, dd), 6.76 (2H, brs), 6.86 (1H, br s), 6.99 (1H, td), 7.08 (2H, d), 7.16 (2H, t),7.25-7.35 (2H, m), 7.76-7.80 (3H, m), 7.89 (2H, d), 8.34 (1H, s) and10.00 (1H, s).

2. From Intermediate (XVc)

To a solution of intermediate (XVc) (19 mg, 0.047 mmol) in DMSO (1.5 mL)was added aq sodium hydroxide (1M, 98 μL, 0.098 mmol). The mixture wasstirred at RT for 10 min and then treated with a solution of tosylate(XIa) (ex APIChem, Catalogue Number: AC-8330, 12.4 mg, 27.6 mmol) inDMSO (0.5 mL). The reaction mixture was stirred at 60° C. for 2 h,cooled to RT and water (10 mL) was added. The resulting mixture wasextracted with EtOAc (3×10 mL) and the combined organic extracts weredried and evaporated in vacuo to afford a brown oil. The crude productso obtained was purified by flash column chromatography (SiO₂, 4 g, 0-2%MeOH in DCM, gradient elution) to afford the beige solid (23 mg). Theproduct was repurified by flash column chromatography (SiO₂, 4.0 g,0-50% EtOAc in DCM, gradient elution) to afford the title compound,Example 1, as an off-white solid (14 mg, 42%); R^(t) 2.60 min (Methoda); m/z 683 (M+H)⁺ (ES⁺).

EXAMPLE 24-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(2,4-difluorophenyl)benzamide

To a suspension of intermediate (IIa) (74.0 mg, 0.125 mmol) in DCM (1.0mL) was added 1-chloro-N,N,2-trimethylprop-1-en-1-amine (50.0 μL, 1.88mmol) and the reaction mixture stirred at RT for 18 hr and thenevaporated in vacuo. The resulting tan coloured solid was dissolved inDCM (1.0 mL) and was added to a solution of 2,4-difluoroaniline (19.0μL, 0.188 mmol) in pyridine (0.5 mL). The reaction was stirred at RT for3 h, then diluted with DCM (3.0 mL) and acidified to pH 2 by theaddition of 1M hydrochloric acid (6.0 mL). The mixture was passedthrough a phase separator and the organics evaporated in vacuo. Thecrude product so obtained was purified by flash column chromatography(SiO₂, 12 g, 50-100% EtOAc in isohexane, gradient elution) to afford thetitle compound, Example 2, as an off-white solid (59.0 mg, 66%); R^(t)2.53 min (Method a); m/z 701 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16(1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.16 (4H, m),3.42-3.45 (4H, m), 3.68 (1H, dd), 3.74-3.80 (2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.06-7.12(3H, m), 7.25-7.36 (3H, m), 7.56 (1H, td), 7.77 (1H, s), 7.89 (2H, d),8.34 (1H, s), 9.81 (1H, s).

EXAMPLE 34-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide

To a suspension of intermediate (IIc) (100 mg, 0.169 mmol), EDCI (64.9mg, 0.339 mmol) and DMAP (1.03 mg, 8.47 μmol) in pyridine (900 μL) wasadded 4-fluoroaniline (17.6 μL, 0.186 mmol). The reaction mixture wasstirred at RT for 18 hr and was then poured into water (50 mL). Theresulting solid was collected by filtration and was washed with water(10 mL) and with diethyl ether (10 mL) and dried in vacuo at 40° C. togive the title compound, Example 3, as an off-white powder (94.0 mg,80%); R^(t) 2.25 min (Method a); m/z 684 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.16(1H, dd), 2.24 (3H, s), 2.37-2.43 (1H, m), 2.52-2.57 (1H, m), 3.39-3.42(4H, m), 3.49-3.51 (4H, m), 3.69 (1H, dd), 3.74-3.78 (2H, m), 4.05 (1H,dd), 4.58 (2H, dd), 6.67 (1H, d), 7.00 (1H, td), 7.07 (2H, d), 7.13-7.22(3H, m), 7.25-7.34 (2H, m), 7.76-7.79 (3H, m), 7.89 (2H, d), 8.34 (1H,s), 10.00 (1H, s).

EXAMPLE 44-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-cyanophenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide

To a suspension of intermediate (IIb) (72.0 mg, 0.120 mmol), EDCI (45.1mg, 0.235 mmol) and DMAP (1.00 mg, 8.19 μmol) in pyridine (1.0 mL) wasadded 4-fluoroaniline (11.1 μL, 0.118 mmol) and the reaction mixturestirred at RT for 72 hr. The mixture was diluted with water (30 mL) andstirred for 5 min. The solid so formed was collected by filtration andpurified by flash column chromatography (SiO₂, 4 g, 0-10% MeOH in DCM,gradient elution) to afford the title compound, Example 4, as a whitesolid (36.8 mg, 44%); R^(t) 2.59 min (Method a); 694 m/z (M+H)⁺ (ES⁺);¹H NMR δ: 2.18 (1H, dd), 2.40-2.46 (1H, m), 2.56-2.64 (1H, m), 3.23-3.26(4H, m), 3.43-3.45 (4H, m), 3.78-3.96 (3H, m), 4.04 (1H, dd), 4.59 (2H,dd), 6.98 (1H, td), 7.08-7.19 (5H, m), 7.25-7.37 (4H, m), 7.74-7.80 (3H,m), 7.89 (2H, d), 8.34 (1H, s), 10.03 (1H, s).

EXAMPLE 54-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-2,5-dimethylphenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide

To a solution of intermediate (XVc) (70.0 mg, 0.167 mmol) in DMSO (1.0mL) at 30° C. was added aq. NaOH (20.0 μL, 12.5M, 0.250 mmol). After 30min the tosylate (XIa) (83.0 mg, 0.184 mmol) was added and the reactionmixture was stirred at 30° C. for 18 hr, then cooled to RT and pouredinto water (15 mL). The resulting precipitate was collected byfiltration, and was washed with water (20 mL) to provide a white solid.The crude product was purified by preparative HPLC (Method 2) to providethe title compound, Example 5 as a white solid (34.0 mg, 29%); R^(t)2.89 min (Method a); m/z 697 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.07 (3H, s), 2.16(1H, dd), 2.25 (3H, s), 2.37-2.43 (1H, s), 2.53-2.57 (1H, m), 2.89-2.92(4H, m), 3.41-3.44 (4H, m), 3.69 (1H, dd), 3.74-3.81 (2H, m), 4.05 (1H,dd), 4.58 (2H, dd), 6.71 (1H, s), 6.88 (1H, s), 7.00 (1H, td), 7.07 (2H,d), 7.16 (2H, t), 7.26-7.35 (2H, m), 7.76-7.80 (3H, m), 7.89 (2H, d),8.35 (1H, s), 10.00 (1H, s).

EXAMPLE 64-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(5-fluoropyridin-2-yl)benzamide

To a suspension of intermediate (IIa) (100 mg, 0.17 mmol), EDCI (65 mg,0.34 mmol) and DMAP (1.04 mg, 8.48 μmol) in pyridine (1.40 mL) was added5-fluoropyridin-2-amine (23.9 mg, 0.25 mmol) and the reaction mixturewas stirred at RT for 15 hr. The mixture was diluted with DCM (8.0 mL)and acidified by the addition of 1M hydrochloric acid (2.0 mL). Themixture was then passed through a phase separator and the organicsevaporated in vacuo. The crude product was purified by preparative HPLC(Method 2) to provide the title compound, Example 6 as a white solid(41.0 mg, 34%); R^(t) 2.51 min (Method a); m/z 684 (M+H)⁺ (ES⁺); ¹H NMRδ: 2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m),3.13-3.16 (4H, m), 3.43-3.46 (4H, m), 3.69 (1H, dd), 3.74-3.80 (2H, m),4.05 (1H, dd), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00(1H, td), 7.05 (2H, d), 7.25-7.34 (2H, m), 7.74-7.79 (2H, m), 7.97 (2H,d), 8.20 (1H, dd), 8.34 (1H, s), 8.37 (1H, d), 10.56 (1H, s).

EXAMPLE 74-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)piperazin-1-yl)-N-(5-cyanopyridin-2-yl)benzamide

A vial charged with intermediate (XXa) (100 mg, 0.170 mmol),6-bromonicotinonitrile (31.1 mg, 0.170 mmol), Xantphos (4.91 mg, 8.49μmol, 5 mol %), tris(dibenzylideneacetone) dipalladium(0) (3.89 mg, 4.25μmol, 2.5 mol %) and cesium carbonate (166 mg, 0.510 mmol) was evacuatedand refilled with nitrogen three times before DMF (1.0 mL) was added.The reaction was heated to 100° C. for 18 hr, then cooled to RT andpartitioned between DCM (15 mL) and brine (20 mL). The phases wereseparated and the organic layer was dried and concentrated in vacuo togive an oily residue which was triturated in methanol (6.0 mL). Theresulting solid was isolated by filtration, washed with diethyl ether(10 mL) and dried in vacuo at 40° C. to give the crude solid. Thetrituration step was repeated with methanol (3.0 mL) and dried in vacuoto give title compound, Example 7, as an off-white powder (71.5 mg,59%); R^(t) 2.71 min (Method b); m/z 691 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.10(3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.15(4H, m), 3.45-3.47 (4H, m), 3.69 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H,t), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.05(2H, d), 7.25-7.34 (2H, m), 7.74-7.77 (2H, m), 7.99 (2H, d), 8.05 (1H,dd), 8.34 (1H, s), 8.48 (1H, dd), 10.92 (1H, s).

The following compound examples (Table 2) may be prepared by similarsynthetic methods to the aforementioned examples or by methods describedelsewhere herein:

TABLE 2 Additional Compound Examples of the Invention Example No.,Structure, Name, Purification Method and Analytical and Spectral Data 8

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(3-cyanophenyl)benzamide. Purified by trituration fromether/DCM; R^(t) 2.53 min (Method a); m/z 690 (M + H)⁺ (ES⁺); 1H NMR δ:2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m),3.13-3.16 (4H, m), 3.44-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m),4.05 (1H, dd), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.01(1H, td), 7.10 (2H, d), 7.26- 7.34 (2H, m), 7.51-7.58 (2H, m), 7.77 (1H,s), 7.91 (2H, d), 8.05 (1H, td), 8.26-8.27 (1H, m), 8.35 (1H, s), 10.27(1H, s). 9

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-cyanophenyl)benzamide. Prep HPLC Method 2; R^(t)2.53 min (Method a); m/z 690 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s),2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.16 (4H, m),3.44-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.09 (2H, d),7.25-7.35 (4H, m), 7.77-7.80 (3H, m), 7.91 (2H, m), 7.97-8.01 (2H, m),8.34 (1H, s), 10.34 (1H, s). 10

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-chlorophenyl)-piperazin-1-yl)-N-(4-cyanophenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.79min (Method a); m/z 710 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.18 (1H, dd),2.39-2.45 (1H, m), 2.53-2.61 (1H, m), 3.20-3.23 (4H, m), 3.45-3.48 (4H,m), 3.75-3.89 (3H, m), 4.05 (1H, dd), 4.59 (2H, dd), 6.93-7.03 (3H, m),7.08-7.12 (3H, m), 7.26-7.33 (2H, m), 7.77-7.82 (3H, m), 7.92 (2H, d),7.98-8.02 (2H, m), 8.34 (1H, s), 10.35 (1H, s). 11

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3,5-dimethylphenyl)-piperazin-1-yl)-N-(4-cyanophenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.70min (Method a); m/z 704 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.09-2.15 (7H, m),2.40-2.46 (1H, m), 2.52-2.58 (1H, m), 3.17-3.20 (4H, m), 3.41-3.45 (5H,m), 3.55 (1H, dd), 3.84 (1H, dd), 4.09 (1H, t), 4.58 (2H, br s), 6.65(2H, br s), 7.01 (1H, td), 7.10 (2H, d), 7.26-7.34 (2H, m), 7.77-7.81(3H, m), 7.91 (2H, d), 7.99 (2H, d), 8.34 (1H, s), 10.35 (1H, s). 12

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-ylmethoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-difluoromethoxyphenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.59min (Method a); m/z 731 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16(1H, dd), 2.37-2.43 (1H, m), 2.52-2.59 (1H, m), 3.12-3.18 (4H, m),3.40-3.46 (4H, m), 3.68 (1H, dd), 3.75-3.79 (2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.08 (2H, d),7.15 (2H, d), 7.25-7.35 (3H, m), 7.77-7.81 (3H, m), 7.90 (2H, d), 8.34(1H, s), 10.03 (1H, s). 13

4-(4-(5-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-(trifluoromethoxy)phenyl)benzamide. Purified bycolumn chromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t)2.83 min (Method a); m/z 749 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s),2.16 (1H, dd), 2.37-2.43 (1H, m), 2.53-2.58 (1H, m), 3.14-3.16 (4H, m),3.42-3.45 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.09 (2H, d),7.25-7.35 (4H, m), 7.77 (1H, s), 7.87-7.91 (4H, m), 8.34 (1H, s), 10.13(1H, s). 14

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)-piperazin-1-yl)-N-(3-fluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 50-100% EtOAc in iso-hexane, gradient elution);R^(t) 2.60 min (Method a); m/z 683 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H,s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.16 (4H,m), 3.43-3.45 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd),4.58 (2H, dd), 6.76 (2H, br s), 6.86-6.91 (2H, br m), 7.00 (1H, td),7.09 (2H, d), 7.25-7.39 (3H, m), 7.54-7.57 (1H, m), 7.74-7.78 (2H, m),7.90 (2H, d), 8.34 (1H, s), 10.12 (1H, s). 15

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3,5-dimethylphenyl)-piperazin-1-yl)-N-(4-fluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.71min (Method a); m/z 697 (M + H)⁺ (ES⁺); 1H NMR δ: 2.13 (1H, dd), 2.15(6H, s), 2.40-2.46 (1H, m), 2.52-2.58 (1H, m), 3.17-3.20 (4H, m),3.40-3.46 (5H, m), 3.56 (1H, dd), 3.84 (1H, t), 4.09 (1H, t), 4.55-4.62(2H, m), 6.66 (2H, s), 7.01 (1H, td), 7.08 (2H, d), 7.17 (2H, t),7.26-7.34 (2H, m), 7.76-7.80 (3H, m), 7.89 (2H, d), 8.34 (1H, s), 10.02(1H, s). 16

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-hydroxymethyl-phenyl)piperazin-1-yl)-N-(4-fluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.21min (Method a); m/z 699 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.16 (1H, dd),2.36-2.42 (1H, m), 2.51-2.57 (1H, m), 3.15-3.17 (4H, m), 3.44-3.46 (4H,m), 3.68 (1H, dd), 3.73-3.80 (2H, m), 4.03 (1H, dd), 4.45 (2H, d), 4.58(2H, d), 4.99 (1H, t), 6.77-6.83 (2H, m), 7.00 (1H, td), 7.07-7.10 (3H,m), 7.15-7.19 (2H, m), 7.26-7.33 (2H, m), 7.76-7.76 (3H, m), 7.89 (2H,d), 8.35 (1H, s), 10.02 (1H, s). 17

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-fluorophenyl)-piperazin-1-yl)-N-(4-fluorophenyl)benzamide. Prep HPLC Method 2; R^(t)12.67 min (Method a); m/z 687 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.14 (1H, dd),2.37-2.43 (1H, m), 2.53-2.58 (1H, m), 3.20-3.22 (4H, m), 3.42-3.44 (4H,m), 3.71-3.84 (3H, m), 4.02 (1H, dd), 4.58 (2H, dd), 6.73 (1H, dd),6.92-7.02 (3H, m), 7.09 (2H, d), 7.17 (2H, t), 7.25-7.32 (2H, m),7.76-7.80 (3H, m), 7.89 (2H, d), 8.34 (1H, s), 10.02 (1H, s). 18

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-chlorophenyl)-piperazin-1-yl)-N-(4-fluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 12.80min (Method a); m/z 703 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.17 (1H, dd),2.38-2.44 (1H, m), 2.53-2.60 (1H, m), 3.19-3.22 (4H, m), 3.42-3.44 (4H,m), 3.74-3.88 (3H, m), 4.04 (1H, dd), 4.59 (2H, dd), 6.92-7.02 (3H, m),7.07-7.09 (3H, m), 7.14-7.19 (2H, m), 7.25-7.32 (2H, m), 7.76-7.80 (3H,m), 7.89 (2H, d), 8.33 (1H, s), 10.01 (1H, s). 19

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-sulfamoylphenyl)benzamide. Prep HPLC Method 3;R^(t) 2.19 min (Method a); m/z 744 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H,s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.14-3.16 (4H,m), 3.44-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.80 (2H, m), 4.05 (1H, dd),4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.09(2H, d), 7.24-7.35 (4H, m), 7.76-7.79 (3H, m), 7.91-7.96 (4H, m), 8.34(1H, s), 10.25 (1H, s). 20

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-(N-methylsulfamoyl)phenyl)benzamide. Purified bycolumn chromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t)2.35 min (Method a); m/z 758 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s),2.16 (1H, dd), 2.37-2.43 (4H, m), 2.52-2.58 (1H, m), 3.14-3.16 (4H, m),3.44-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.10 (2H, d),7.25- 7.35 (3H, m), 7.71-7.75 (2H, m), 7.77 (1H, s), 7.92 (2H, d),7.98-8.01 (2H, m), 8.34 (1H, s), 10.29 (1H, s). 21

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)-piperazin-1-yl)-N-(4-(N, N-dimethylsulfamoylphenyl)benzamide. Purifiedby column chromatography (SiO₂, 50-100% EtOAc in isohexane, gradientelution); R^(t) 2.52 min (Method a); m/z 772 (M + H)⁺ (ES⁺); ¹H NMR δ:2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.60 (7H, m),3.14-3.16 (4H, m), 3.43-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m),4.05 (1H, dd), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00(1H, td), 7.10 (2H, d), 7.25- 7.35 (2H, m), 7.71 (2H, d), 7.77 (1H, s),7.92 (2H, d), 8.05 (2H, d), 8.34 (1H, s), 10.34 (1H, s). 22

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-fluorophenyl)-piperazin-1-yl)-N-(4-sulfamoylphenyl)benzamide. Purified by columnchromatography (SiO₂, 50-100% EtOAc in isohexane, then 5-10% MeOH (1%NH₃) in DCM, gradient elution); 1H NMR δ: 2.15 (1H, dd), 2.37-2.43 (1H,m), 2.53-2.58 (1H, m), 3.20-3.22 (4H, m), 3.44-3.46 (4H, m), 3.72-3.78(2H, m), 3.83 (1H, dd), 4.03 (1H, dd), 4.58 (2H, dd), 6.73 (1H, br d),6.91-7.02 (3H, m), 7.10 (2H, d), 7.24-7.32 (4H, m), 7.76 (1H, s), 7.78(2H, d), 7.91-7.96 (4H, m), 8.33 (1H, s), 10.25 (1H, s). 23

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-cyano-2-fluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.61min (Method a); m/z 708 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16(1H, dd), 2.37-2.42 (1H, m), 2.52-2.58 (1H, m), 3.13-3.15 (4H, m),3.44-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79(2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.09 (2H, d),7.26- 7.34 (2H, m), 7.70 (1H, dd), 7.77 (1H, s), 7.89-7.98 (4H, m), 8.35(1H, s), 10.10 (1H, s). 24

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-fluorophenyl)-piperazin-1-yl)-N-(4-cyano-2-fluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 50-100% EtOAc in isohexane, gradient elution);R^(t) 2.69 min (Method a); m/z 712 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.14 (1H,dd), 2.37-2.43 (1H, m), 2.53-2.57 (1H, m), 3.20-3.22 (4H, m), 3.44-3.47(4H, m), 3.72-3.84 (3H, m), 4.03 (1H, dd), 4.58 (2H, dd), 6.73 (1H, dd),6.91-7.02 (3H, m), 7.09 (2H, d), 7.25-7.32 (2H, m), 7.70 (1H, dd), 7.76(1H, s), 7.89-7.98 (4H, m), 8.33 (1H, s), 10.08 (1H, s). 25

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-(difluoromethoxy)-3-fluorophenyl)benzamide.Purified by column chromatography (SiO₂, 0-4% MeOH in DCM, gradientelution); R^(t) 2.51 min (Method a); m/z 749 (M + H)⁺ (ES⁺); ¹H NMR δ:2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.53-2.58 (1H, m),3.12-3.18 (4H, m), 3.41-3.47 (4H, m), 3.68 (1H, dd), 3.74-3.78 (2H, m),4.05 (1H, t), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 6.97-7.02(1H, m), 7.09 (2H, d), 7.17-7.35 (4H, m), 7.58 (1H, d), 7.77 (1H, s),7.89-7.94 (3H, m), 8.34 (1H, s), 10.18 (1H, s). 26

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl-N-(2,5-difluorophenyhbenzamide. Purified by columnchromatography (SiO₂, 0-3% MeOH in DCM, gradient elution); R^(t) 2.67min (Method a); m/z 701 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.12 (3H, s), 2.17(1H, dd), 2.38-2.43 (1H, m), 2.54-2.60 (1H, m), 3.25-3.43 (4H, m),3.53-3.62 (4H, m), 3.71- 3.83 (3H, m), 4.05 (1H, dd), 4.58 (2H, dd),6.86 (1H, br s), 6.98-7.12 (5H, m), 7.27-7.37 (4H, m), 7.56-7.61(1H, m),7.79 (1H, s), 7.91 (2H, d), 8.37 (1H, s), 9.93 (1H, s). 27

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl-N-(3,4-difluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.65min (Method a); m/z 701 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16(1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.16 (4H, m),3.43-3.45(4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.08 (2H, d),7.25- 7.43 (3H, m), 7.52-7.56 (1H, m), 7.77 (1H, s), 7.89 (2H, d), 7.95(1H, ddd), 8.34 (1H, s), 10.14 (1H, s). 28

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)-piperazin-1-yl)-N-(3,5-difluorophenyl)benzamide. Prep HPLC Method 1;R^(t) 2.73 min (Method a); m/z 701 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H,s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.57 (1H, m), 3.13-3.16 (4H,m), 3.43-3.46 (4H, m), 3.68 (1H, m), 3.74-3.79 (2H, m), 4.05 (1H, dd),4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 6.90 (1H tt), 7.00 (1H,td), 7.09 (2H, d), 7.25-7.35 (2H, m), 7.55-7.58 (2H, m), 7.77 (1H, s),7.89 (2H, d), 8.34 (1H, s), 10.26 (1H, s). 29

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-chloro-2-fluorophenyl)benzamide. Prep HPLC Method1; Rt 2.74 min (Method a); m/z 717 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H,s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.16 (4H,m), 3.43-3.45 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd),4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.07(2H, d), 7.25- 7.35 (3H, m), 7.51 (1H, dd), 7.63 (1H, t), 7.77 (1H, s),7.89 (2H, d), 8.34 (1H, s), 9.87 (1H, s). 30

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl-N-(4-chloro-3-fluorophenyl)benzamide. Purified byprecipitation with water, filtration and washing with water; R^(t) 2.80min (Method a); m/z 717 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16(1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.16 (4H, m),3.43-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58(2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.09 (2H, d),7.25- 7.35 (2H, m), 7.53 (1H, t), 7.61 (1H, dd), 7.77 (1H, s), 7.89 (2H,d), 7.97 (1H, dd), 8.34 (1H, s), 10.23 (1H, s). 31

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)-piperazin-1-yl)-N-(2,4,6-trifluorophenyl)benzamide. Purified by columnchromatography (SiO₂, 50-100% EtOAc in isohexane, gradient elution);R^(t) 2.48 min (Method a); m/z 719 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H,s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.57 (1H, m), 2.92 (2H, t),3.13-3.16 (4H, m), 3.43-3.45 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m),4.05 (1H, t), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H,td), 7.09 (2H, d), 7.26-7.34 (2H, m), 7.77 (1H, s), 7.89 (2H, d), 8.34(1H, s), 9.79 (1H, s). 32

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-fluorophenyl)-3-methylbenzamide. Purified by columnchromatography (SiO₂, 0-4% MeOH in DCM, gradient elution); R^(t) 2.58min (Method a); m/z 697 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.17(1H, dd), 2.35 (3H, s), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.05-3.07(4H, m), 3.17-3.19 (4H, m), 3.69 (1H, dd), 3.75-3.80 (2H, m), 4.06 (1H,dd), 4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td),7.14-7.20 (3H, m), 7.25-7.35 (2H, m), 7.76-7.80 (5H, m), 8.34 (1H, s),10.12 (1H, s). 33

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-fluorophenyl)-2-methylbenzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.57min (Method a); m/z 697 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16(1H, dd), 2.37-2.43 (4H, m), 2.52-2.58 (1H, m), 3.13-3.15 (4H, m),3.34-3.37 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, t), 4.58(2H, dd), 6.76 (2H, br s), 6.85-6.90 (3H, m), 7.00 (1H, td), 7.13-7.17(2H, m), 7.25-7.35 (2H, m), 7.40 (1H, d), 7.72-7.77 (3H, m), 8.34 (1H,s), 10.09 (1H, s). 34

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)-piperazin-1-yl)-N-(4-fluorophenyl)-3-methoxybenzamide. Purified bycolumn chromatography (SiO₂, 50-100% Et0Ac in isohexane, gradientelution); R^(t) 2.43 min (Method a); m/z 713 (M + H)⁺ (ES⁺); ¹H NMR δ:2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.56 (1H, m),3.14-3.22 (8H, m), 3.68 (1H, dd), 3.75-3.80 (2H, m), 3.89 (3H, s), 4.05(1H, dd), 4.58 (2H, dd), 6.75 (2H, br s), 6.85 (1H, br s), 6.97-7.03(2H, m), 7.19 (2H, t), 7.25- 7.35 (2H, m), 7.51 (1H, d), 7.59 (1H, dd),7.75-7.79 (3H, m), 8.34 (1H, s), 10.10 (1H, s). 35

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-fluorophenyl)-2-methoxybenzamide. Purified bycolumn chromatography (SiO₂, 0-4% MeOH in DCM, gradient elution); R^(t)2.47 min (Method a); m/z 713 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.10 (3H, s),2.16 (1H, dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.13-3.16 (4H, m),3.44-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 3.99 (3H, s), 4.05(1H, dd), 4.58 (2H, dd), 6.65 (1H, d), 6.69 (1H, dd), 6.76 (2H, br s),6.86 (1H, br s), 7.00 (1H, td), 7.16 (2H, t), 7.25-7.35 (2H, m),7.73-7.77 (4H, m), 8.34 (1H, s), 9.88 (1H, s). 36

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)3-methylphenyl)-piperazin-1-yl)-N-(6-cyanopyridin-2-yl)benzamide. Purified bytrituration from MeOH; R^(t) 2.75 min (Method a); m/z 691 (M + H)⁺(ES⁺); ¹H NMR δ: 2.10 (3H, s), 2.16 (1H, dd), 2.37-2.42 (1H, m),2.52-2.58 (1H, m), 3.12-3.14 (4H, m), 3.44-3.47 (4H, m), 3.67 (1H, dd),3.74-378 (2H, m), 4.04 (1H, t), 4.58 (2H, dd), 6.75 (2H, br s), 6.86(1H, br s), 6.97-7.06 (3H, m), 7.26-7.34 (2H, m), 7.75-7.77 (2H, m),7.99 (2H, d), 8.05 (1H, dd), 8.35 (1H, s), 8.48 (1H, dd), 10.94 (1H, s).37

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(4-cyanopyridin-2-yl)benzamide. Purified byprecipitation from MeOH; R^(t) 2.72 min (Method b); m/z 691 (M + H)⁺(ES⁺); 1H NMR δ: 2.10 (3H, s), 2.16 (1H, dd), 2.37-2.43 (1H, m),2.52-2.58 (1H, m), 3.13-3.15 (4H, m), 3.45-3.47 (4H, m), 3.68 (1H, dd),3.74-3.79 (2H, m), 4.05 (1H, t), 4.58 (2H, dd), 6.76 (2H, br s), 6.86(1H, br s), 7.00 (1H, td), 7.06 (2H, d), 7.25- 7.34 (2H, m), 7.57 (1H,dd), 7.77 (1H, s), 7.99 (2H, d), 8.34 (1H, s), 8.51 (1H, br s), 8.62(1H, dd), 10.92 (1H, s). 38

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-hydroxymethylphenyl)-piperazin-1-yl)-N-(5-cyanopyridin-2-yl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.19min (Method a); m/z 707 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.16 (1H, dd),2.37-2.42 (1H, m), 2.52-2.57 (1H, m), 3.14-3.17 (4H, m), 3.47-3.49 (4H,m), 3.67-3.80 (3H, m), 4.04 (1H, dd), 4.45 (2H, d), 4.58 (2H, dd), 4.97(1H, t), 6.77-6.83 (2H, m), 6.99 (1H, td), 7.05-7.08 (3H, m), 7.25-7.33(2H, m), 7.77 (1H, s), 7.99 (2H, d), 8.27 (1H, dd), 8.34-8.37 (2H, m),8.84 (1H, dd), 10.99 (1H, s). 39

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl-N-(5-(trifluoromethoxy)pyridin-2-yl)benzamide Purified bycolumn chromatography (SiO₂, 0-100% EtOAc in DCM, gradient elution);R^(t) 2.82 min (Method a); m/z 750 (M + H)⁺ (ES⁺); ¹H NMR δ 2.10 (3H,s), 2.16 (1H, dd), 2.37-2.42 (1H, m), 2.53-2.58 (1H, m), 3.13-3.15 (4H,m), 3.44-3.46 (4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd),4.58 (2H, dd), 6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.06(2H, d), 7.25-7.34 (2H, m), 7.77 (1H, s), 7.93 (1H, dd), 7.98 (2H, d),8.31 (1H, d), 8.34 (1H, s), 8.47 (1H, d), 10.74 (1H, s). 40

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-fluorophenyl)-piperazin-1-yl)-N-(5-fluoropyridin-2-yl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.63min (Method a); m/z 688 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.14 (1H, dd),2.37-2.43 (1H, m), 2.53-2.59 (1H, m), 3.19-3.21 (4H, m), 3.43-3.45 (4H,m), 3.72-3.84 (3H, m), 4.03 (1H, dd), 4.58 (2H, d), 6.73 (1H, dd),6.90-7.02 (3H, m), 7.05 (2H, d), 7.25-7.31 (2H, m), 7.74-7.79 (2H, m),7.97 (2H, d), 8.21 (1H, dd), 8.33 (1H, s), 8.37 (1H, d), 10.57 (1H, s).41

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-3-methylphenyl)-piperazin-1-yl)-N-(5-chloropyridin-2-yl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.72min (Method a); m/z 701 (M + H)⁺ (ES⁺); ¹H NMR δ 2.10 (3H, s), 2.16 (1H,dd), 2.37-2.43 (1H, m), 2.52-2.58 (1H, m), 3.12-3.15 (4H, m), 3.43-3.46(4H, m), 3.68 (1H, dd), 3.74-3.79 (2H, m), 4.05 (1H, dd), 4.58 (2H, dd),6.76 (2H, br s), 6.86 (1H, br s), 7.00 (1H, td), 7.05 (2H, d), 7.26-7.34(2H, m), 7.77 (1H, s), 7.93 (1H, dd), 7.97 (2H, d), 8.23 (1H, dd), 8.34(1H, s), 8.42 (1H, dd), 10.66 (1H, s). 42

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)6-methylpyridin-2-yl)-piperazin-1-yl)-N-(5-cyanopyridin-2-yl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.18min (Method a); m/z 692 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.15 (1H, dd), 2.24(3H, s), 2.37-2.43 (1H, m), 2.52-2.57 (1H, m), 3.44-3.50 (8H, m), 3.68(1H, dd), 3.74-3.78 (2H, m), 4.04 (1H, dd), 4.58 (2H, dd), 6.67 (1H, d),6.97-7.06 (3H, m), 7.21 (1H, d), 7.25-7.34 (2H, m), 7.77 (1H, s), 7.99(2H, d), 8.26 (1H, dd), 8.34-8.36 (2H, m), 8.84 (1H, dd), 10.98 (1H, s).43

4-(4-(4-(((3R,5R)-5-((1H-1,2,4-triazol-1-yl)methyl)-5-(2,4-difluorophenyl)tetrahydrofuran-3-yl)methoxy)-6-methylpyridin-2-yl)-piperazin-1-yl)-N-(5-fluoropyridin-2-yl)benzamide. Purified by columnchromatography (SiO₂, 0-10% MeOH in DCM, gradient elution); R^(t) 2.16min (Method a); m/z 685 (M + H)⁺ (ES⁺); ¹H NMR δ: 2.16 (1H, dd), 2.24(3H, s), 2.37-2.43 (1H, m), 2.54-2.58 (1H, m), 3.41-3.43 (4H, m),3.48-3.50 (4H, m), 3.68 (1H, dd), 3.74-3.78 (2H, m), 4.04 (1H, dd), 4.58(2H, dd), 6.67 (1H, d), 6.97-7.05 (3H, m), 7.21 (1H, d), 7.25-7.34 (2H,m), 7.74- 7.79 (2H, m), 7.97 (2H, d), 8.21 (1H, dd), 8.34 (1H, s), 8.37(1H, d), 10.57 (1H, s).

Biological Testing: Experimental Methods

Assessment of Planktonic Fungus Growth

a. Resazurin-Microtitre Assay

This assay was conducted using a modified, published method (Monteiro etal., 2012). Spores of Aspergillus fumigatus (NCPF2010, Public HealthEngland, Wiltshire) were cultured in Sabouraud dextrose agar for 3 days.A stock spore suspension was prepared from a Sabouraud dextrose agarculture by washing with PBS-tween (10 mL; PBS containing 0.05% Tween-20,100 U/mL Penicillin and 100 U/mL Streptomycin). The spore count wasassessed using a Neubauer haemocytometer and, using PBS adjusted to 10⁶spores/mL. A working suspension of spores (10⁴ spores/mL) was preparedin filter sterilised MOPS RPMI-1640 (50 mL; RPMI-1640 containing 2 mML-glutamine, 2% glucose and 0.165 M MOPS, buffered to pH 7 with NaOH).Resazurin sodium salt (100 μL of 1% solution; Sigma-Aldrich, Dorset, UK)was added to the spore suspension and mixed well. The sporesuspension-resazurin mixture (100 μL/well) was added to 384-well plates(Catalogue number 353962, BD Falcon, Oxford, UK).

Simultaneously, test compounds (0.5 μL DMSO solution) were added to 100μL of the spore-resazurin mixture in quadruplicate to provide a finalDMSO solution of 0.5% using an Integra VIAFLO 96 (Intergra, Zizers,Switzerland). For non-spore control wells, MOPS-RPMI-resazurin solution(100 μL) was added instead of the spore-resazurin mixture. The plate wascovered with a Breathe Easier membrane (Catalogue No Z763624,Sigma-Aldrich, Dorset, UK), and incubated (35° C., 5% CO₂) untilfluorescence in the inoculated wells was double that of the controlwells (around 24 hr). The fluorescence of each well (545 nm(excitation)/590 nm (emission), gain 800, focal height 5.5 mm) wasdetermined using a multi-scanner (Clariostar: BMG, Buckinghamshire, UK).The percentage inhibition for each well was calculated and the MIC₅₀,MIC₇₅ and MIC₉₀ values were calculated from the concentration-responsecurve generated for each test compound.

b. Broth Microdilution Assay

This assay was conducted using a modified method published by EUCAST(Rodriguez-Tudela et al., 2008). Spores of Aspergillus fumigatus(NCPF2010, NCPF7010 (Methionine 220 mutation), NCPF7099 (Glycine G54mutation) from Public Health England, Wiltshire; TR34/L98H mutants fromSt Louis Hospital, Paris, France) were cultured in Sabouraud dextroseagar for 3 days. A stock spore suspension was prepared from a Sabourauddextrose agar culture by washing with PBS-tween (10 mL; PBS containing0.05% Tween-20, 100 U/mL Penicillin and 100 U/mL Streptomycin). Thespore count was assessed using a Neubauer haemocytometer and thenadjusted to 10⁶ spores/mL with PBS. A working suspension of spores(2×10⁵ spores/mL) was prepared in filter sterilised, BSA MOPS RPMI-1640(50 mL; RPMI-1640 containing 2 mM L-glutamine, 0.5% BSA, 2% glucose,0.165 M MOPS, buffered to pH 7 with NaOH). For the assay, BSA MOPSRPMI-1640 (50 μL/well) was added throughout the 384-well plate(Catalogue number 353962, BD Falcon, Oxford, UK) first. Test compounds(0.5 μL DMSO solution) were then added in quadruplicate using an IntegraVIAFLO 96 (Integra, Zizers, Switzerland), and mixed well using a platemixer. Subsequently 50 μL of the working spore suspension prepared abovewas added to all wells except non-spore control wells. For non-sporecontrol wells, BSA MOPS-RPMI solution (50 μL/well) was added instead.The plate was covered with a plastic lid, and incubated (35° C. withambient air) for 48 hr. The OD of each well at 530 nm was determinedusing a multi-scanner (Clariostar: BMG, Buckinghamshire, UK). Thepercentage inhibition for each well was calculated and the MIC₅₀, MIC₇₅and MIC₉₀ values were calculated from the concentration-response curvegenerated for each test compound.

Fungus panel screening was conducted by Eurofins Panlabs Inc. The MICand MIC₅₀ values of the test articles were determined following theguidelines of the Clinical and Laboratory Standards Institute, brothmicrodilution methods for yeast (CLSI M27-A2), (CLSI, 2002) and forfilamentous fungi (CLSI M38-A), (CLSI, 2008).

Aspergillus fumigatus Infection of Bronchial Epithelial Cells

BEAS2B cells were seeded in 96-well plates (100 μL; 30,000 cells/well;Catalogue No 3596, Sigma Aldrich, Dorset, UK) in 10% FBS RPMI-1640 andwere then incubated (37° C., 5% CO₂) for one day before experimentation.Test compounds (0.5 μL DMSO solution) or vehicle (DMSO) were added toeach well to give a final DMSO concentration of 0.5%. BEAS2B cells wereincubated with test compounds for 1 hr (35° C., 5% CO₂) before infectionwith Aspergillus fumigatus (20 μL; Public Health England) conidiasuspension (0.5×10⁵/ml in 10% FBS RPMI-1640). The plate was incubatedfor 24 hr (35° C., 5% CO₂). Supernatant (50 μL) was collected andtransferred to a PCR plate (Catalogue No L1402-9700, Starlab, MiltonKeynes, UK), which was frozen (−20° C.) until use. After thawing,supernatant (5 μL) was diluted 1:20 by adding R7-PBS solution (95 μL;1:4 R7 to PBS; Bio-Rad Laboratories, Redmond, Wash., USA). GM levels inthese samples (50 μL) were measured using Platelia GM-EIA kits (Bio-RadLaboratories, Redmond, Wash., USA). The percentage inhibition for eachwell was calculated and the IC₅₀ value was calculated from theconcentration-response curve generated for each test compound.

Aspergillus fumigatus Infection of Human Alveoli Bilayers

In vitro models of human alveoli, consisting of a bilayer of humanalveolar epithelial cells and endothelial cells, were prepared aspreviously described (Hope et al., 2007). This system allowsadministration of a test compound to the upper (“air” space) and/orlower (“systemic” space) compartments. This flexibility has beenexploited to explore the effects of combination treatments by dosingcompound Example 1 to the upper chamber and posaconazole or otheranti-fungal agents to the lower chamber. Primary human pulmonary arteryendothelial cells (HPAEC) were harvested and diluted to 10⁶ cells/mL inEGM-2 media (Lonza, Basel, Switzerland). Transwells were inverted andthe cell suspension (100 μL/well) was applied to the base of eachtranswell. The inverted transwells were incubated at RT within a flowhood for 2 hr after which they were turned upright. EGM-2 media wasadded to the lower (700 μL/well) and upper (100 μL/well) compartmentsand the transwells were incubated for 48 hr (37° C., 5% CO₂). The EGM-2media in the lower compartment was then replaced with fresh EGM-2 media.A549 cells were harvested and diluted to 5×10⁵ cells/mL in 10% EBM, thenadded to the upper compartment (100 μL/well) of all transwells and theplates incubated for 72 hr (37° C., 5% CO₂). Conidia of Aspergillusfumigatus (the itraconazole sensitive strain NCPF2010 and theitraconazole resistant strain TR34-L98H) were cultured separately inSabouraud dextrose agar for 3 days. A stock conidia suspension of eitherstrain was prepared from a Sabouraud dextrose agar culture by washingwith PBS-tween (10 mL; PBS containing 0.05% Tween-20, 100 U/mLPenicillin and 100 U/mL Streptomycin). The conidia count was assessedusing a Neubauer haemocytometer and adjusted to 10⁶ conidia/mL with PBS.A working stock of conidia was prepared in EBM (conc of 10⁵ conidia/mL)immediately prior to use.

Test and reference compounds (or neat DMSO as the vehicle) were added tothe appropriate wells of 24-well plates (3 μL/well containing 600 μL of2% FBS EBM) for lower compartment treatment and to 96-well plates (1μL/well containing 200 μL of 2% FBS EBM) for the treatment of the uppercompartment, to provide a final DMSO concentration of 0.5%. The media inthe upper compartment was aspirated and that containing the appropriatetest and reference compounds, or vehicle, were added (100 μL/well).Transwells were then transferred into the 24-well plate containing thetest and reference compounds or DMSO vehicle. After incubation for 1 hr(35° C., 5% CO₂) the conidia suspension (10 μL/well) was added to theupper compartment of each transwell. Plates were then incubated for 24hr (35° C., 5% CO₂). Supernatants from each compartment (5μL/compartment) were collected and stored (−20° C.). Media was replaceddaily after collection of the supernatants and all wells were treatedwith test and reference compounds or with DMSO, as described above, for3 days. Samples continued to be collected until fungal growth wasvisible by eye in all transwells. The levels of GM in the supernatant inlower compartment were then measured by ELISA (BioRad, CA, USA) as anindex of Aspergillus fumigatus invasion.

Cell Viability: Resazurin Assay

BEAS2B cells were seeded in 384-well plates (100 μL; 3000/well/; BDFalcon, Catalogue No 353962) in RPMI-LHC8 (RPMI-1640 and LHC8 mediacombined in equal proportions) one day before experimentation. Forcell-free control wells, RPMI-LHC8 (100 μL) was added. Test compounds(0.5 μL of a DMSO solution) were added to give a final DMSOconcentration of 0.5% using an Integra VIAFLO 96 (Integra, Zizers,Switzerland). BEAS2B cells were incubated with each test compound for 1day (37° C./5% CO₂ in RPMI-LHC8). After addition of resazurin stocksolution (5 μL, 0.04%) the plates were incubated for a further 4 hr (37°C./5% CO₂). The fluorescence of each well at 545 nm (excitation) and 590nm (emission) was determined using a multi-scanner (Clariostar: BMGLabtech). The percentage loss of cell viability was calculated for eachwell relative to vehicle (0.5% DMSO) treatment. Where appropriate, aCC₅₀ value was calculated from the concentration-response curvegenerated from the concentration-response curve for each test compound.

In Vivo Anti-Fungal Activity

Aspergillus fumigatus (ATCC 13073 [strain: NIH 5233], American TypeCulture Collection, Manassas, Va., USA) was grown on Malt agar (NissuiPharmaceutical, Tokyo, Japan) plates for 6-7 days at RT (24±1° C.).Spores were aseptically dislodged from the agar plates and suspended insterile distilled water with 0.05% Tween 80 and 0.1% agar. On the day ofinfection, spore counts were assessed by haemocytometer and the inoculumwas adjusted to obtain a concentration of 1.67×10⁸ spores mL⁻¹ ofphysiological saline.

To induce immunosuppression and neutropenia, A/J mice (males, 5 weeksold) were dosed with hydrocortisone (Sigma H4881; 125 mg/kg, sc,) ondays 3, 2 and 1 before infection, and with cyclophosphamide (SigmaC0768; 250 mg/kg, ip) 2 days before infection. On day 0, animals wereinfected with the spore suspension (35 μL intra-nasally).

Test compounds were administered intra-nasally (35 μL of a suspension of0.08-2.00 mg/mL in physiological saline) once daily, 30 min beforeinfection on day 0 and then on days 1, 2 and 3 (representingprophylactic treatment) or on days 1, 2 and 3 only (representingtherapeutic treatment). For extended prophylactic treatment, testcompounds (35 μL of a suspension of 0.0032 or 0.016 mg/mL inphysiological saline) were administered intra-nasally once daily forseven days; then 30 min before infection on day 0, and thereafter,either on days 1, 2 and 3 after infection, or on day 0 only. The effectsof these treatment paradigms were compared with those obtained whentreatment was restricted to one day and 30 min before inoculation andthen on days 1, 2 and 3 post infection; or reduced still further to oneday and 30 min before infection only. Animal body weights were monitoreddaily and those exhibiting a reduction ≥20%, compared with their bodyweight on day 0, were culled.

Six hours after the last dose, animals were anesthetised, the tracheawas cannulated and BALF was collected. The total number of alveolarcells was determined using a haemocytometer, and the numbers of alveolarmacrophages and neutrophils were determined by FACS analysis (EPICS®ALTRA II, Beckman Coulter, Inc., Fullerton, Calif., USA) usinganti-mouse MOMA2-FITC (macrophage) or anti-mouse 7/4 (neutrophil),respectively, as previously reported (Kimura et al., 2013). The levelsof IFN-γ and IL-17 in BALF, and IL-6 and TNFα in serum were determinedusing Quantikine® mouse IFN-γ, IL-17, IL-6 or TNF-α ELISA kit (R&Dsystems, Inc., Minneapolis, Minn., USA) respectively. MDA, an oxidativestress marker, was assayed using OxiSelect® TBARS Assay Kits (MDAQuantitation; Cell Biolabs Inc, San Diego, Calif., USA). Aspergillus GMin serum was determination using Platelia GM-EIA kits (Bio-RadLaboratories, Redmond, Wash., USA). Cut-off index was calculated by theformula: Cut-off index=OD in sample/OD in cut-off control provided inkit. For tissue fungal load assays, 100 mg of lung tissue was removedaseptically and homogenized in 0.2 mL of 0.1% agar in sterile distilledwater. Serially diluted lung homogenates were plated on Malt agar plates(50 μL/plate), and incubated at 24±1° C. for 72 to 96 h. The colonies ofA. fumigatus on each plate was counted and the fungal titre presented asCFU per gram of lung tissue.

Severely immunosuppressed, neutropenic A/J mice (males, 5 weeks old),which had been dosed with hydrocortisone (Sigma H4881; 125 mg/kg, sc,)daily for three days before infection and with cyclophosphamide (SigmaC0768; 250 mg/kg, ip) two days before infection were used to evaluatethe effects of the combined treatment of compound Example (I)administered intranasally and posaconazole dosed orally. On day 0,animals were infected intranasally with 35 μL of the spore suspension(1.67×10⁸ spores/mL in physiological saline) of Aspergillus fumigatus(ATCC 13073 [strain: NIH 5233]). Compound Example (I), prepared as asuspension in isotonic saline (0.4 mg/mL), was dosed once daily by anintra-nasal injection (35 μL/mouse) on days 1-6 after infection.Posaconazole (1 mg/kg) was given orally once daily on days 1-6 afterinfection. Body weight and survival were monitored daily up to day 7.

Summary of Screening Results

The compounds of the invention, as disclosed herein, demonstrate potentinhibitory activity against infection of bronchial epithelial cells byazole sensitive Aspergillus fumigatus and, where tested, fungal growth,as evaluated by the resazurin assay (Table 3). With a single exception,incubation with the compounds of the invention had no or little effecton the viability of BEAS2B bronchial epithelial cells at concentrationsup to, at least, 10 μM. Particularly, in these assay systems compoundExample 1 showed significantly greater potency than voriconazole andamphotericin B and similar potency to posaconazole.

TABLE 3 The effects of treatment with Voriconazole, Posaconazole,Amphotericin B and the compound examples of the invention on Aspergillusfumigatus (NCPF2010) planktonic fungal growth, on fungal infection ofBEAS2B bronchial epithelial cells and on BEAS2B cell viability.MIC₅₀/MIC₇₅/CC₅₀ Values in assay system indicated (nM) TreatmentPlanktonic fungal Infection of BEAS2B (Test Compound growth¹ BEAS2Bcells² Cell Viability³ Example No.) MIC₅₀ MIC₇₅ MIC₅₀ CC₅₀ Voriconazole90.8 168 154 >28600 Posaconazole 3.64 6.94 4.48 >14300 Amphotericin B28.5 64.4 nt 977 1 1.98 5.02 5.43 >12200 2 0.74 3.06 1.42 >14300 3 nt nt51.7 >14600 4 nt nt 7.77 >14400 5 nt nt 123 >14400 6 nt nt 23.9 >14600 7nt nt 12.5 >14500 8 nt nt 14.2 548 9 nt nt 5.21 >14500 10 nt nt14.5 >14100 11 nt nt 284 >14200 12 nt nt 97.3 >13700 13 4.27 17.823.9 >13400 14 nt nt 8.36 >14600 15 nt nt 7.64 >14400 16 nt nt0.82 >14300 17 nt nt nt >14600 18 nt nt 86.6 >14200 19 nt nt 17.2 >1340020 nt nt 36.1 >13200 21 nt nt 3.86 >13000 22 nt nt 4.77 >13400 23 nt nt3.37 >14100 24 nt nt nt >14100 25 nt nt 230 >13400 26 nt nt >1430 >1430027 nt nt nt >14300 28 nt nt 11.7 >14300 29 nt nt 14.5 >13900 30 nt nt1050 >13900 31 nt nt 10.7 >13900 32 nt nt 26.1 >14400 33 nt nt43.5 >14400 34 nt nt 21.9 >14000 35 nt nt 125 >14000 36 nt nt3.52 >14500 37 nt nt 18.8 >14500 38 nt nt 4.86 >14100 39 nt nt nt >1330040 nt nt 6.87 >14500 41 nt nt nt >14300 42 nt nt nt >14500 43 nt nt2.85 >14600 Table Footnotes: ¹Resazurin-microtitre assay; ²Bronchialepithelial cells; ³n = 3-5;

Furthermore, the compounds of the invention exhibit potent inhibitoryactivity against planktonic fungal growth as evaluated in a brothmicrodilution assay (Table 4). In this assay, the compounds of theinvention commonly showed significantly greater potency versus theposaconazole-resistant strains (NCPF7099, NCPF7100 and TR34/L98H) aswell as a posaconazole-sensitive strain (NCPF2010) than didposaconazole, voriconazole and Amphotericin B.

TABLE 4 The effects of treatment with Voriconazole, Posaconazole,Amphotericin B and the compound examples of the invention on planktonicfungal growth of isolates of Aspergillus fumigatus. Treatment MIC₇₅Values (nM) against the (Test Compound indicted Aspergillus fumigatusisolates¹ Example No.) NCPF2010 NCPF7099 NCPF7100 L98H Voriconazole 49696.7 596 >2860 Posaconazole 15.3 112 71.5 150 Amphotericin B 382365 >1080 209 1 13.6 16.5 19.7 56.7 2 7.00 28.3 21.3 81.6 3 18.0 27.645.1 33 4 8.68 41.3 38.3 73.7 5 13.3 157 96.6 162 6 12.5 21.2 21.4 131 75.86 14.1 4.07 41.9 8 12.7 >1450 >1450 >1450 9 29.1 7.39 23.2 114 1022.0 9.96 29.5 81.2 11 23.6 49.0 52.9 179 12 13.4 22.8 30.3 45.9 13 25.333.0 32.4 101 14 43.1 33.2 41.0 73.5 15 29.0 18.4 8.45 >1440 16 5.129.26 6.58 33.5 17 2.15 18.5 13.3 409 18 22.2 11.0 47.1 73.3 19 16.6 8.9022.7 57.7 20 18.8 11.1 14.6 46.1 21 37.4 21.1 31.7 51.7 22 11.3 16.422.0 >1340 23 12.6 28.4 44.3 60.2 24 9.90 20.5 14.0 191 25 84.5 25.147.6 65.9 26 31.1 131 91.0 126 27 19.3 12.7 22.3 136 28 31.9 26.6 36.1198 29 28.5 37.0 53.8 120 30 58.4 34.7 41.3 191 31 43.9 10.9 67.9 163 3269.7 48.1 60.9 69.5 33 13.5 48.9 23.6 67.9 34 44.6 29.0 14.8 73.9 3563.4 109 111 178 36 41.0 44.5 34.6 134 37 28.4 24.6 22.0 194 38 12.431.9 33.6 65.4 39 52.0 nt nt 65.9 40 12.5 24.0 14.6 73.1 41 57.6 nt nt261 42 46.8 30.1 40.6 80.2 43 23.7 32.3 34.3 56.4 Table Footnotes:¹Broth microdilution assay, n = 3

The effects of compound Example 1 on the growth of wide range of fungalpathogens were evaluated using the CLSI broth microdilution methods.Compound Example 1 was found to be a potent inhibitor of the growth ofRhizopus oryzae, Cryptococcus neoformans, Chaetomimum globosum,Penicillium chrysogenum and Trichophyton rubrum as well as some CandidaSpp (Table 5).

TABLE 5 The effects of compound Example 1 on the growth of a range offungi species. Example 1 Voriconazole Posaconazole Fungal MIC₅₀ MIC₁₀₀MIC₅₀ MIC₁₀₀ MIC₅₀ MIC₁₀₀ Agent Strain (μg/mL) (μg/mL) (μg/mL)Aspergillus ATCC204304 1.0 >8.0 1.0 2.0 0.063 0.13 flavus AspergillusATCC9348 >8.0 >8.0 >8.0 >8.0 0.25 1.0 pullulans Candida 20240.0470.031 >8.0 0.031 >8.0 0.031 >8.0 albicans ATCC10231 0.13 >8.0 0.25 >8.00.13 >8.0 20183.073 0.5 >8.0 4.0 >8.0 0.25 >8.020186.025 >8.0 >8.0 >8.0 >8.0 >8.0 >8.0 Candida ATCC36583 0.5 >8.00.25 >8.0 0.5 >8.0 glabrata R363 0.5 >8.0 >8.0 >8.0 0.5 >8.0 RhizopusATCC11145 0.063 2.0 8.0 >8.0 0.13 >8.0 oryzae Cryptococcus ATCC240670.008 1.0 0.016 1.0 0.016 0.25 neoformans Chaectomium ATCC446990.063 >8.0 0.5 1.0 0.13 0.25 globosum Penicillium ATCC9480 0.031 >8.01.0 2.0 0.063 0.13 chrysogenum Trichophyton ATCC10218 <0.008 0.031<0.008 0.063 <0.008 0.031 rubrum Table Footnotes: MIC₅₀/MIC₁₀₀ =concentration required for 50% and 100% inhibition of fungal growth byvisual inspection (CLSI).

Monotherapy with either compound Example 1 (0.1 μg/mL in the upperchamber) or posaconazole (0.01 μg/mL in the lower chamber) inhibited GMproduction on day 1 in human alveoli bilayers. However, the inhibitoryeffects of these treatments were lost rapidly thereafter (Table 6). Incontrast, combination treatment of compound Example 1 with posaconazoleshowed sustained inhibition of invasion post infection. Consequently,the DFB₅₀ for the combination treatment was 5.48 days, much longer thanthe values for either compound alone.

This synergistic or additive effect of combination therapy was alsoconfirmed when treatment with compound Example 1 was combined with thatof intraconazole, voriconazole or caspofungin (results not shown).

TABLE 6 Effects of compound Example 1, Posaconazole and the treatmentcombination on Aspergillus fumigatus (NCPF2010) invasion into the lowerchamber in human alveoli bilayers (transwells). GM Levels in the LowerChamber for Treatments Indicated OD value (% inhibition vs.control)lTreatment Example 1¹ Posaconazole² Combination Day Vehicle Upper ChamberLower Chamber Treatment 0 0 0 0 0 1 0.68 0.091 (86)   0.064 (91)   0.007(99)  2 1.19 1.15 (3.4) 1.01 (15)  0.011 (99)  3 1.19 1.14 (3.7) 1.14(4.1) 0.025 (98)  4 1.18 1.13 (4.5) 1.17 (1.1) 0.11 (91) 5 1.18 1.18(0.3)   1.18 (−0.6) 0.42 (64) 6 1.18   1.18 (−0.3)   1.19 (−1.1) 0.73(38) 7 1.18 1.16 (0.9) 1.17 (0.3)  1.15 (2.0) 8 1.16 1.13 (2.8) 1.15(0.8)  1.12 (3.7) DFB₅₀ Values for 1.13 1.45 5.48 treatments indicatedTable Footnotes: ¹Dosed at 0.1 μg/mL; ²Dosed at 0.01 μg/mL; DFB₅₀: Daystaken to reach a fungal burden of 50% of control

In addition, this combination treatment has been tested in bilayersinfected with the azole resistant strain of Aspergillus fumigatus:TR34-L98H. (Table 7) Monotherapy with compound Example 1 (1 μg/mL) inthe upper chamber or with posaconazole (0.1 μg/mL) in the lower chambershowed limited benefit. In contrast, the combination of compound Example1 and posaconazole showed marked inhibitory effects on fungal invasioninto the lower chamber. The beneficial effect of the combinationtreatment was observed on day 1 post infection, but disappeared afterday 2.

TABLE 7 Effects of compound Example 1, Posaconazole and the treatmentcombination on Aspergillus fumigatus (TR34-L98H strain) invasion intothe lower chamber in the alveolar bilayer cell system (transwells). GMLevels in the Lower Chamber for Treatments Indicated OD value (%inhibition vs.control)l Compound Example 1¹ Posaconazole² CombinationTreatment Day Upper Chamber Lower Chamber Treatment 0 0 0 0 1 0.35 0.039(88)     0.013 (96) 2 0.99 1.02 (−2.7) 0.082 (92) 3 0.99 0.97 (1.7)   0.54 (45) 4 1.01 1.02 (−1.4)     1.09 (−8.8) DFB₅₀ 1.10 1.64 2.93Values for treatments indicated Table Footnotes: ¹Dosed at 1 μg/mL;²Dosed at 0.1 μg/mL; DFB₅₀: Days taken to reach a fungal burden of 50%of control

When given intranasally to immunocompromised, neutropenic mice, on day 0and days 1-3 following inoculation (prophylactic treatment) in ahead-to-head comparison, compound Example 1 showed superior effects toposaconazole on reducing body weight loss, measured over 3 days, causedby infection with Aspergillus fumigatus. (Table 8).

TABLE 8 Comparison of the effects of treatment with compound Example 1and Posaconazole on the body weight loss of immunocompromised,neutropenic mice caused by infection with Aspergillus fumigatus. Bodyweight loss caused by infection with A. fumigatus ² Drug (% Inhibitionof weight loss) Treatment¹ Day 1 Day 2 Day 3 Vehicle plus Spores 9.2 ±1.5   14.3 ± 1.9   19.3 ± 1.4   Posaconazole 7.3 ± 2.0 (21) 13.4 ± 1.9(6)  18.1 ± 2.0 (6)  Example 1 6.1 ± 1.8 (34)  8.7 ± 2.5 (39) 11.1 ± 5.6(42) Table Footnotes: ¹Dosed at 0.4 mg/mL intra-nasally; ²% weight losscompared with animal weight on day 0.

Furthermore, prophylactic and therapeutic treatment with compoundExample 1 showed superior effects to posaconazole on fungal load in thelung, as well as on GM concentrations in both BALF and serum, postinfection. The data for this compound, used in prophylactic andtherapeutic dosing regimens, are shown in Table 9 and FIGS. 1, 2 and 3(ID₅₀ values presented in Table 10).

TABLE 9 The effects of prophylactic and therapeutic treatment withcompound Example 1 on CFU in lung and galactomannan concentrations inthe BALF and serum of Aspergillus fumigatus infected,immuno-compromised, neutropenic mice. % Inhibition of response TreatmentDrug Conc CFU GM in BALF GM in serum Regimen (mg/mL) (/mg of lung) (COI)(COI) Vehicle None 28.4 ± 16.9   4.8 ± 0.40   5.3 ± 1.1   plus SporesCompound 0.08 15.2 ± 13.7 (46) 0.70 ± 0.39 (85) 0.81 ± 0.52 (85) Example1: 0.4 2.1 ± 1.6 (93) 0.37 ± 0.46 (92) 0.24 ± 0.18 (95) Prophylactic 20.8 ± 0.7 (97) 0.13 ± 0.02 (97) 0.18 ± 0.07 (97) Treatment Compound 0.43.8 ± 1.0 (87) 0.24 ± 0.06 (95) 0.29 ± 0.11 (95) Example 1: 2 1.9 ± 1.7(93) 0.22 ± 0.14 (95) 0.25 ± 0.19 (95) Therapeutic 10 0.5 ± 0.3 (98)0.11 ± 0.05 (98) 0.24 ± 0.11 (95) Treatment Table Footnotes: The datafor fungal load are shown as the mean ± standard error of the mean (SEM;n = 5-6).

TABLE 10 The ID₅₀ values for prophylactic treatment with Posaconazoleand compound Example 1 on fungal load in the lung and on galactomannanconcentrations in the BALF and serum, of Aspergillus fumigatus infected,immuno-compromised, neutropenic mice. Drug substance (Prophylactic ID₅₀Values for response indicated (mg/mL) Treatment) Lung Fungal Load GM inBALF GM in serum Compound 0.086 <0.08 <0.08 Example 1 Posaconazole 0.241.3 0.47

In addition, prophylactic or therapeutic treatment with certain othercompounds of the invention, showed superior effects to posaconazole onfungal load in the lung, as well as on GM concentrations in both BALFand serum, post infection. The data from these studies are shown below(Tables 11 and 12).

TABLE 11 The effects of prophylactic treatment (on days 0, 1, 2 and 3)with test compounds on fungal load in the lung and galactomannanconcentrations in the BALF and serum of Aspergillus fumigatus infected,immuno- compromised, neutropenic mice. Prophylactic Treatment Drug %Inhibition versus Infected Controls Compound Conc Lung GM in GM inExample No. (mg/mL) Fungal Load BALF serum Posaconazole 0.4 44 20 34 10.4 81 92 95 2 0.4 25 43 27 4 0.4 85 84 90 13 0.4 18 49 32 17 0.4 83 4572 19 0.4 62 16 30 19 2 74 41 47 23 0.4 56 71 79

TABLE 12 The effects of therapeutic treatment (on days 1, 2 and 3) withtest compounds on fungal load in the lung and galactomannanconcentrations in both BALF and serum, in Aspergillus fumigatusinfected, immuno- compromised, neutropenic mice. Therapeutic TreatmentDrug % Inhibition versus Infected Control Compound Conc Lung GM in GM inExample No. (mg/mL) Fungal Load BALF serum 1 0.4 97 94 91 3 0.08 98 9592 3 0.4 96 86 89 16 0.08 91 76 62 16 0.4 96 88 85

Prophylactic treatment with compound Example 1, also inhibitedinflammatory cell accumulation in BALF (Table 13), in a similar fashionto posaconazole. In addition, prophylactic treatment with compoundExample 1 showed superior inhibitory effects to posaconazole versusIL-17, IFNγ and MDA concentrations in BALF, and the comparative ID₅₀values for compound Example 1 and for posaconazole in independentexperiments are displayed in Table 14.

TABLE 13 The effects of prophylactic and therapeutic treatment withcompound Example 1 on macrophage and neutrophil accumulation into theBALF of Aspergillus fumigatus infected, immunocompromised, neutropenicmice. Cell numbers in BALF × 10⁵/mL Drug Conc (% inhibition) Treatment(mg/mL) Macrophage Neutrophil Vehicle plus 0.65 ± 0.14   0.49 ± 0.09  Spores Compound 0.08 0.40 ± 0.15 (38) 0.37 ± 0.04 (24) Example 1 0.40.32 ± 0.07 (51) 0.26 ± 0.12 (47) Prophylactic 2 0.26 ± 0.05 (60) 0.22 ±0.04 (55) Treatment Compound 0.4 0.43 ± 0.05 (34) 0.38 ± 0.04 (22)Example 1 2 0.40 ± 0.11 (38) 0.34 ± 0.05 (31) Therapeutic 10 0.32 ± 0.07(51) 0.27 ± 0.08 (45) Treatment Table Footnotes: The data for cellnumber are shown as the mean ± standard error of the mean (SEM), N =5-6.

TABLE 14 The ID₅₀ values for prophylactic treatment with Posaconazoleand compound Example 1 on IL-17, IFNγ and MDA levels in the BALF ofAspergillus fumigatus infected, immuno-compromised, neutropenic mice.Drug substance ID₅₀ Values for biomarkers (Prophylactic indicated(mg/mL) Treatment) IL-17 IFNγ MDA Compound 0.074 <0.08 0.11 Example 1Posaconazole 0.61 0.22 0.69

Furthermore, data showing the effects of compound Example 1 on IFNγ,IL-17 and MDA levels in the BALF, when administered eitherprophylactically or therapeutically, are shown in Table 15 and theeffects on serum, IL-6 and TNFα are shown in Table 16.

TABLE 15 The effects of prophylactic and therapeutic treatment withcompound Example 1 on IFNγ, IL-17 and MDA levels in the BALF ofAspergillus fumigatus infected, immunocompromised, neutropenic mice.Biomarker Concentrations in BALF Drug (% Inhibition) Treatment Conc IFNγIL-17 MDA Regimen (mg/mL) (pg/mL) (pg/mL) (pg/mL) Vehicle 9.2 ± 1.0  19.8 ± 3.6    1.8 ± 0.2   plus Spores Compound 0.08 3.7 ± 1.7 (60) 9.8 ±5.3 (51) 0.96 ± 0.32 (47) Example 1 0.4 3.0 ± 0.8 (67) 6.7 ± 4.9 (66)0.57 ± 0.22 (68) Prophylactic 2 2.5 ± 0.3 (73) 3.2 ± 0.8 (84) 0.34 ±0.05 (81) Treatment Compound 0.4 4.3 ± 2.2 (53) 8.5 ± 2.9 (57) 0.45 ±0.10 (75) Example 1 2 3.3 ± 0.8 (64) 4.0 ± 0.8 (80) 0.37 ± 0.10 (79)Therapeutic 10 2.1 ± 0.3 (77) 2.9 ± 0.7 (85) 0.25 ± 0.05 (86) TreatmentTable Footnotes: The data for biomarker concentrations are shown as themean ± standard error of the mean (SEM), N = 5-6.

TABLE 16 The effects of prophylactic and therapeutic treatment withcompound Example 1 on IL-6 and TNFα levels in the serum of Aspergillusfumigatus infected, immunocompromised, neutropenic mice Conc of Biomarkers (pg/mL) Treatment Drug Conc (% Inhibition) Regimen (mg/mL) IL-6TNFα Vehicle 284 ± 112   25.6 ± 8.0    plus Spores Compound 0.08  159 ±73.3 (44) 11.8 ± 5.9 (54)  Example 1 0.4 86.3 ± 46.9 (70) 7.3 ± 3.5 (71)Prophylactic 2 44.5 ± 12.2 (84) 4.7 ± 0.4 (82) Treatment Compound 0.451.7 ± 16.8 (82) 6.2 ± 0.5 (76) Example 1 2 44.2 ± 11.4 (84) 5.5 ± 0.7(79) Therapeutic 10 35.9 ± 10.4 (87) 4.9 ± 0.6 (81) Treatment TableFootnotes: The data for biomarker concentrations are shown as the mean ±standard error of the mean (SEM), N = 5-6.

Therapeutic treatment with compound Example 1 was also found to maintainpotent inhibition of lung fungal load, serum galactomannan levels and onBALF cytokine concentrations in Aspergillus fumigatus infected,immunocompromised, neutropenic mice. (Tables 8, 9, 10 and 13; and FIGS.1, 2 and 3).

The effects of extended prophylactic dosing with compound Example 1 onbiomarkers in Aspergillus fumigatus infected, immuno-compromised,neutropenic mice were also evaluated. Extended prophylaxis with thecompound of Example 1 was found to inhibit fungal load in the lung, aswell as the GM concentrations in both BALF and serum, at 25 fold lowerdoses than those used in a previous biomarker study (Table 17).Furthermore, the data suggest an accumulation of anti-fungal effects inthe lung on repeat dosing since seven days of prophylaxis producedgreater anti-fungal effects than did prophylactic treatment for a singleadded day. The compound's persistence of action in the lung is suggestedby the finding that treatment on days −7 to day 0 generated superioranti-fungal effects on day 3 than those resulting from treatment on days−1 and 0, only. Nevertheless this abbreviated dosing protocol was stillprotective.

TABLE 17 The effects of extended prophylactic dosing of compound Example1 on fungal load (CFU) in lung and GM concentrations in the BALF andserum of Aspergillus fumigatus infected, immuno-compromised, neutropenicmice. Dose of Treatment Compound Values and % Inhibition of response³Regimen¹ Example 1 CFU GM in BALF GM in Serum (Days dosed) (μg/mL) (/mgof lung) (COI) (COI) Vehicle plus None 34.7 ± 10.7   5.1 ± 0.9    4.3 ±1.0    Spores² −7 to +3 3.2 8.3 ± 2.0 (76) 2.6 ± 0.36 (49) 1.8 ± 0.43(58) −1 to +3 3.2 9.5 ± 3.3 (73) 2.8 ± 0.71 (45) 2.2 ± 0.69 (49) −7 to+3 16 5.0 ± 2.3 (86) 1.7 ± 0.39 (67) 1.4 ± 0.20 (67) −1 to +3 16 6.1 ±2.8 (82) 2.2 ± 0.61 (57) 1.6 ± 0.41 (63) −7 to 0   16 6.7 ± 1.7 (81) 2.3± 0.52 (55) 1.7 ± 0.59 (60) −1, 0 16 13.1 ± 2.6 (62)  4.5 ± 0.50 (12)4.0 ± 0.88 (7)  Table Footnotes: ¹The N value was six for all drugtreated groups; ²The N value was five for the vehicle treated group;³The data for fungal load and GM levels are shown as the mean ± standarderror of the mean and the percentage inhibition, with respect tovehicle.

The influence on survival of combining the treatments of compoundExample 1, dosed topically, with oral Posaconazole, was evaluated inseverely immuno-compromised, neutropenic mice after inoculation withAspergillus fumigatus. Monotherapy with compound Example 1 (0.4 mg/mL,given intranasally) or with Posaconazole (1.0 mg/kg, dosed orally)showed only a very limited therapeutic benefit. In contrast, thecombination of compound Example 1 and Posaconazole demonstrated a markedincrease on survival time following infection (Table 18).

TABLE 18 Effects of compound Example 1 and Posaconazole as monotherapyand in combination on survival in severely immune-compromised,neutropenic mice infected with Aspergillus fumigatus. No. of MedianLog-rank test Treatment survivors survival for survival Regimen Dose(Route) on day 7 (%) (days) (vs.infection) Vehicle none 0/6 (0) 5 —Compound 0.4 mg/mL (in) 0/6 (0) 6 p < 0.05  Example 1 Posaconazole 1mg/kg, (po)  0/6 (0) 6.5 Not significant Compound 0.4 mg/mL (in)  5/6(83) Undefined p < 0.001 Example 1 plus 1 mg/kg (po) Posaconazole TableFootnotes: N = 8 per group.

In Vivo Pharmacokinetics

It is a commonly used procedure for pulmonary, therapeutic agents to bedosed into the lungs of animals, for example mice, and plasma collectedat various time points after dosing in order to characterise theresulting systemic exposure to the administered compound. The compoundof the invention may be tested in such in vivo systems.

Summary of the Biological Profile of the Compound Examples of theInvention.

The compound examples of the invention disclosed herein have been foundto be potent inhibitors of bronchial epithelial cell infection byAspergillus fumigatus and of planktonic growth. The compounds of theinvention also inhibited the growth of posaconazole-resistant andvoriconazole-resistant Aspergillus fumigatus isolates, commonlydemonstrating greater potency than posaconazole, voriconazole andintraconazole against these strains. In vivo, in Aspergillus fumigatusinfected, immunocompromised, neutropenic mice, compounds of theinvention, demonstrated potent inhibition of Aspergillus fumigatusinfection, whether dosed prophylactically or therapeutically. Inparticular compound Example 1 demonstrated potent inhibition ofAspergillus fumigatus infection-associated lung immune responses whetherdosed prophylactically or as a treatment. In addition, compound Example1 was highly efficacious in reducing infection-dependent body weightloss. These inhibitory effects were superior to those of posaconazole.It is significant that the beneficial anti-fungal effects of thecompound of Example 1 and the compounds of the invention generally areobserved in both a prophylactic and a therapeutic dosing setting.

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Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

The invention claimed is:
 1. A process for preparing a compound offormula (II):

wherein: R² represents hydrogen, halo, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy orC₁₋₄ haloalkoxy; R³ represents halo, cyano, C₁₋₄ alkyl, or C₁₋₄hydroxyalkyl; R⁴ represents hydrogen or C₁₋₄ alkyl; R^(a) represents H;and X represents CH or N; or a salt thereof; which comprises reacting acompound of formula (VIII):

wherein: R³ represents, halo, cyano, C₁₋₄alkyl, or C₁₋₄hydroxyalkyl; R⁴represents, hydrogen or C₁₋₄alkyl; and X represents CH or N; or a saltthereof; with a compound of formula (XIV):

wherein R² represents hydrogen, halo, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy orC₁₋₄ haloalkoxy; and R^(a) represents C₁₋₅ alkyl; or a salt thereof; togive a compound of formula (IV)

wherein R² represents hydrogen, halo, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy orC₁₋₄ haloalkoxy; R³ represents halo, cyano, C₁₋₄ alkyl, or C₁₋₄hydroxyalkyl; R⁴ represents hydrogen or C₁₋₄ alkyl; X represents CH orN; and R^(a) represents C₁₋₅ alkyl; or a salt thereof; followed byhydrolysis of the compound of formula (IV) to give said compound offormula (II).
 2. The process according to claim 1 wherein R² representsH, Me or OMe.
 3. The process according to claim 2 wherein R² representsH.
 4. The process according to claim 1 wherein R³ represents Me, CN, Cl,CH₂ OH or F.
 5. The process according to claim 4 wherein R³ representsMe.
 6. The process according to claim 1 wherein R⁴ represents H or Me.7. The process according to claim 6 wherein R⁴ represents H.
 8. Theprocess according to claim 1 wherein R⁴ represents H and R³ representsMe, CN, Cl, CH ₂ OH or F.
 9. The process according to claim 8 wherein R⁴represents H and R³ represents Me.
 10. The process according to claim 1wherein R⁴ represents Me and R³ represents Me.
 11. The process accordingto claim 1 wherein R² is located ortho to the nitrogen of thepiperazinyl substituent.
 12. The process according to claim 1 wherein R²is located meta to the nitrogen of the piperazinyl substituent.
 13. Theprocess according to claim 1 wherein R⁴ is located ortho to the oxygenof the ether substituent.
 14. The process according to claim 1 wherein Xrepresents CH.
 15. The process according to claim 1 wherein R^(a) offormula (XIV) and formula (IV) represents Me, Et or t-Bu.
 16. Theprocess according to claim 15 wherein R^(a) of formula (XIV) and formula(IV) represents Me.
 17. The process according to claim 15 wherein R^(a)of formula (XIV) and formula (IV) represents t-Bu.
 18. The processaccording to claim 1, wherein the hydrolysis step comprises treatment ofthe compound of formula (IV) with an inorganic base in an aqueousmixture.
 19. The process according to claim 18, wherein the inorganicbase is lithium hydroxide.
 20. The process according to claim 18,wherein the aqueous mixture is a mixture of DMSO and water.